RNA-UNY - The Hudson Valley RNA Club [PDF]

RNA-UNY Structure, Function, Application 2010 Oct 8–9, 2010 Rensselaerville, NY

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RNA-UNY Structure, Function, Application 2010 Oct 8–9, 2010 Rensselaerville, NY

Hosted by the Hudson Valley RNA Club and the Rochester RNA Cluster in partnership with Bioconnex http://www.hudsonvalleyrnaclub.org

Support provided by:

Organizing committee: Marlene Belfort, Wadsworth Center, University at Albany Joan Curcio, Wadsworth Center, University at Albany Alain Laederach, Wadsworth Center, University at Albany Nilesh Banavali, Wadsworth Center, University at Albany David Shub, University at Albany

Scott Tenenbaum, CNSE - University at Albany Carla Theimer, University at Albany Michael Zuker, Rensselaer Polytechnic Institute Yi-Tao Yu, University of Rochester Tom Eickbush, University of Rochester

RNA-UNY: Structure, Function, Application 2010 October 8-9, 2010 Rensselaerville, NY FRIDAY, October 8, 2010 11:30 am

Registration, poster setup and lunch buffet

02:00 pm

Introductions (Marlene Belfort and David Shub)

Session I:

Big RNAs (Chairperson: Scott Tenenbaum) Page 1

02:30 pm

Lynne Maquat (Keynote Talk) mRNP rearrangements during the pioneer round of translation, nonsense-mediated mRNA decay, and thereafter

03:30 pm

Roopa Thapar ERI mediated decay (EMD): Novel exonucleases and pathways in mRNA decay

2

03:50 pm

William Bauer Structural comparison of pseudouridine-modified U2 snRNA/branchpoint site containing RNA duplexes

3

04:10 pm

Leonard Lipovich 4 Genome-wide computational and experimental analysis of long non- coding RNA genes in a complex human brain disorder

04:30 pm

Poster session 1, Wine and Cheese

05:30 pm

Dinner and room check-in

Session II:

Aptamers, Ribozymes and RNAs (Chairperson: Tom Eickbush)

07:00 pm

Daiying Xu 5 Two classes of RNA aptamers as antagonists of human estrogen receptor alpha

07:20 pm

Elisa Biondi A double-site acting kinase ribozyme

6

07:40 pm

Toby Soper Positive regulation of RpoS by small RNAs and the role of Hfq

7

08:00 pm

Larry Gold (Keynote Talk) SOMAmers: Proteomics to lung cancer to a Wellness Chip

8

09:00 pm

Social hour

SATURDAY, October 9, 2010 07:30 am

Breakfast and poster set-up

Session III: RNA Folding and Assembly (Chairperson: Yi-Tao Yu) Page 08:30 am

08:50 am

09:10 am

Joerg Schlatterer Mapping the competing barriers that define alternative RNA folding pathways

9

Steve Meisburger Time-resolved SAXS reconstructions reveal a kinetic intermediate in RNA folding

10

Nathan Napper Alternative assembly pathways of ribosomal 30S subunits in thermophilic bacteria

11

09:30 am

Rob Knight (Mini-Keynote Talk) Nucleotides that are essential but not conserved

10:00 am

Poster session 2

12

Session IV: RNA Structure (Chairperson: Michael Zuker) 11:00 am

Matthew Seetin Automated RNA tertiary structure prediction

13

11:20 am

Jerome Waldispuhl 14 RNAmutants: A computational framework to explore the mutational landscape of structural RNAs: theory and applications

11:40 am

Dan Fabris Higher-order structure of RNA by MS-based approaches

12:00 pm

Anna Pyle (Keynote Talk) 16 The tertiary structure and folding pathways of group II introns: paradigms for the assembly of large, multidomain RNAs and RNPs

01:00 pm

Lunch/departure

15

mRNP Rearrangements During the Pioneer Round of Translation, Nonsense-Mediated mRNA Decay, and Thereafter Jungwook Hwang1, Hanae Sato1, Chenguang Gong1, Yalan Tang1 and Lynne E. Maquat1 1

Department of Biochemistry and Biophysics and the Center for RNA Biology, School of Medicine and Dentistry, 601 Elmwood Avenue, Box 712, University of Rochester, Rochester, New York, 14642, USA In mammalian cells, two different messenger ribonucleoproteins (mRNPs) serve as templates for protein synthesis. Newly synthesized CBP80/CBP20-bound mRNPs initially undergo a pioneer round of translation (Maquat et al., 2010). One purpose of this round of translation is to ensure the quality of gene expression, as exemplified by nonsense-mediated mRNA decay (NMD). NMD largely functions to eliminate mRNAs that prematurely terminate translation, although NMD also contributes to proper gene control, and it targets CBP80/CBP20-bound mRNPs (for recent publications, see Sato et al., 2008; Isken et al., 2008). CBP80/CBP20-bound mRNPs are remodeled to eIF4Ebound mRNPs as a consequence of the pioneer round of translation as well as independently of translation (Sato and Maquat, 2009). eIF4E-bound mRNPs support the bulk of cellular protein synthesis and are the primary targets of mRNA decay mechanisms that conditionally regulate gene expression, such as Staufen1-mediated mRNA decay (see, e.g., Gong et al., 2009). Mechanistic aspects of NMD will be discussed, including how CBP80, which is acquired by the 5’ caps of newly synthesized transcripts within nuclei, promotes NMD at multiple steps by promoting specific mRNP rearrangements (Hwang et al., 2010). Gong, C., Kim, Y.K., Woeller, C.F., Tang, Y. and Maquat, L.E. (2009) SMD and NMD are competitive pathways that contribute to myogenesis: Effects on PAX3 and myogenin mRNAs. Genes & Dev. 23:54-66. Hwang, H., Sato, H., Tang, Y., Matsuda, D. and Maquat, L.E. (2010) UPF1 association with the cap-binding protein, CBP80, promotes nonsense-mediated mRNA decay at two distinct steps. Mol. Cell 39:396-409. Isken, O., Kim, Y.K., Hosoda, N., Mayeur, G.L., Hershey, J.W.B. and Maquat, L.E. (2008) Upf 1 phosphorylation triggers translational repression during nonsense-mediated mRNA decay. Cell 133:314-327. Maquat, L.E., Tarn, Y.-W. and Isken, O. (2010) Specialized features and functions of the pioneer round of translation. Cell, 142:368-374. Sato, H., Hosoda, N. and Maquat, L.E. (2008) Efficiency of the pioneer round of translation affects the cellular site of nonsense-mediated mRNA decay. Mol. Cell 29:255262. Sato, H. and Maquat, L.E. (2009) Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β Genes & Dev. 23:2537-50.

1

ERI Mediated Decay (EMD): Novel exonucleases and pathways in mRNA decay Nithya Krishnan, Anand Parekh, Patrick Itotia, MingJing Wu, and Roopa Thapar* Hauptman-Woodward Medical Research Institute and Department of Structural Biology, SUNY, Buffalo, NY 14203 The ERI family of nucleases are involved in a spectrum of protein-protein and protein-nucleic acid interactions and have been implicated in an array of cellular processes: histone mRNA metabolism, RNAi, 5.8S rRNA processing, and miRNA mediated translational repression and/or decay. ERI-2 or Snipper (in Drosophila) are uncharacterized members of the DEDDh subfamily of exonucleases. We recently published that Snipper1 is a highly active 3’→5’ exonuclease in vitro and can act on both linear and double-stranded RNA substrates. A biochemical property shared by the ERI family of nucleases is that they prefer doublestranded RNA substrates with short 3’ overhangs of 2-5 nucleotides, reminiscent of siRNAs/miRNAs or stem-loop structures. Ongoing studies in our laboratory indicate that hERI-2 may be a RNA-Induced Silencing Complex (RISC) associated nuclease that regulates polyA binding protein (PABP)-dependent mRNA turnover by promoting micro-RNA mediated mRNA decay. hERI-2 interacts directly with PABPC1 in vitro and in vivo. hERI-2 also co-localizes with Ago2 but is not present in P-bodies. Microarray analysis of hERI-2 siRNA treated cells shows that it is involved in regulating the stability of a number of cell cycle regulators, chemokines and cytokines, particularly those with AU-rich elements in their 3’ untranslated regions. Three mRNAs implicated in disease states that are targeted by hERI-2 are the cyclin inhibitor and tumor suppressor CDKN1A, the chemokine CCL5, and the cytokine IL-8. ERI-2 also regulates the cell cycle. Ongoing studies on both ERI-1 and ERI-2 to understand the functional and structural roles of these nucleases in mRNA decay of specific mRNA targets will be presented. 1. Kupsco, J.M., Wu, M.J., Marzluff, W.F., Thapar, R., and Duronio, R.J. Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3′hExo/ERI-1 family of 3′ to 5′ exonucleases. RNA (2006), 12(12): 2103.

2

Structural Comparison of Pseudouridine-Modified U2 snRNA / Branchpoint Site Containing RNA Duplexes W. J. Bauer, S. D. Kennedy, and C.L. Kielkopf Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 Splicing of pre-mRNA is an essential process catalyzed by a spliceosome composed of >100 proteins and five small nuclear (sn)RNAs. A duplex between a consensus pre-mRNA site, called the branchpoint sequence (BPS), and a pseudouridine-modified region of the U2 snRNA contributes to positioning a conserved adenosine within the BPS into an extrahelical position for nucleophilic attack in the first catalytic step of pre-mRNA splicing. Based on previous NMR analysis of an RNA duplex containing S. cerevisiae U2 snRNA/BPS consensus sequences [Newby et al. (2002) NSMB 9:958], it was proposed that the U2 snRNA pseudouridine promotes the extrahelical conformation of a specific branchsite adenosine. However, our crystal structures of pseudouridine-modified U2 snRNA/BPS-containing RNA duplexes demonstrate the capacity of either the branchsite adenosine or a preceding purine nucleotide to assume a bulged conformation [Lin & Kielkopf (2007) Biochemistry 47:5503]. Although this latter observation is consistent with biochemical evidence that either of the two adjacent purines may shift into the bulged position [Query et al. (1994) Genes Dev. 8:587], the viewpoint of a pseudouridine-induced adenosine bulge has continued to dominate the field. By NMR analysis of (i) a duplex containing a pseudouridylated U2 snRNA and a mammalian BPS we here provide evidence for an intrahelical conformation of the 'branchsite' adenosine and a bulged conformation for the adjacent purine in solution. We further characterize the role of pseudouridines in branchsite selection by comparing this structure to BPScontaining duplexes with either (ii) an unmodified U2 snRNA or (iii) a U2 snRNA containing a second pseudouridine.

3

Genome-wide computational and experimental analysis of long non-coding RNA genes in a complex human brain disorder Leonard Lipovich, Becky Cai, Hui Jia, Fabien Dachet, Jeffrey Loeb Center for Molecular Medicine and Genetics, School of Medicine, Wayne State University, 540 East Canfield Street, 3228 Scott Hall, Detroit, MI 48201 Corresponding author: Leonard Lipovich, telephone (313) 577-9683, fax (313) 577-5218, e-mail [email protected] In the noncoding-RNA field, the biology of microRNAs, rRNAs, tRNAs, and sn/snoRNAs typically receives much of the attention. However, transcriptome databases suggest that long non-coding RNA (lncRNA) molecules are more prevalent than microRNAs, and as abundant as mRNAs, in mammalian transcriptional output. Aside from select systems such as X-inactivation and telomerase, mammalian lncRNAs remain poorly understood. To build a systems understanding of human lncRNAs, we annotated human transcript-to-genome alignments supported by public cDNA sequences, constructed a manually curated catalog of over 6,000 lncRNAs, and developed custom microarrays to profile the lncRNA transcriptome. With custom lncRNA, and conventional mRNA, microarrays, we examined brain gene expression in human epilepsy patients, identifying over 300 lncRNAs differentially expressed in surgically resected epileptic and control neocortical regions. We stratified these lncRNAs by their cis-regulatory potential, predicted from their genomic positional relationships with known protein-coding genes. One of the lncRNAs was BDNFOS, transcribed antisense to the BDNF (brain-derived neurotrophic factor) gene. BDNF is a key contributor to epileptogenesis. In human SH-SY5Y neuroblastoma cells, we observed depolarization-dependent BDNFOS suppression and BDNF activation, paralleling microarray results from epileptic patient tissues. To test whether BDNFOS affects, and is not merely coregulated with, BDNF mRNA, we electroporated BDNFOS-targeting siRNAs into SH-SY5Y cells. Upon RNAi-mediated BDNFOS suppression, BDNF mRNA levels increased, consistent with the hypothesis that the lncRNA BDNFOS may regulate BDNF. Our studies establish a precedent for combining bioinformatics, microarrays, and system perturbations to discern the regulatory roles of lncRNAs, but underscore a need for mechanistic understanding of lncRNA functions.

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Two classes of RNA aptamers as antagonists of human estrogen receptor alpha Daiying Xu1, Antonis Kourtidis2, Douglas Conklin2, Hua Shi1 1

SUNY, University at Albany, Department of Biological Sciences, 1400 Washington Avenue, Albany, NY 12222; 2 Gen*NY*Sis Center for Excellence in Cancer Genomics, SUNY, University at Albany, Department of Biomedical Sciences, 1 Discovery Dr., Rensselaer, NY, 12144 Phone: (518) 591-8840; Fax: (518) 442-4767; Email: [email protected] The human estrogen receptor (hER) alpha mediates the growth-stimulating effects of estrogens in a significant number of breast cancers, and is antagonized using small molecule drugs in the treatment of breast cancer. It is composed of multiple domains and bears multiple sites interacting with other factors or elements. However, all hER antagonists currently in use target the ligand-binding pocket of ER. We hypothesized that some other sites on hER alpha may be validated as new drug targets for treating breast cancer and other estrogenopathies. To test this hypothesis, we used RNA aptamers as a means for modulation of hER alpha functions through specific protein surface occlusion. We employed the method of in vitro selection to generate RNA aptamers against the unliganded full-length hER alpha. Two classes of high affinity RNA aptamers with distinct binding properties have been identified. Class I aptamers bind to both isoforms of hER, alpha and beta, with similar affinities; whereas class II aptamer only binds to hER alpha with high affinity. These results suggest that the two classes bind to different sites on hER alpha. To study the efficacy of the aptamers in breast cancer cell lines, genetic systems have been constructed to produce aptamers through transcription from synthetic genes delivered into the cells. In ER-positive breast cancer cell line MCF 7, the expressed aptamers reduced hER alpha-driven luciferase gene activity by 30-50%.

5

A Double-Site Acting Kinase Ribozyme E. Biondi1 and D.H. Burke1 1

Department of Molecular Microbiology and Department of Biochemistry, University of Missouri-Columbia, 415 Life Sciences Center, 1201 E. Rollins St., Columbia, MO – 65211 Elisa Biondi, T. 573-884-5159, F. 573-884-9676, e-mail: [email protected] Our long-term goal is to understand the catalytic potential of RNA, the feasibility of RNA-based evolution in an RNA World, and the possibility of using RNA to engineer artificial gene regulation and metabolism. A key constraint in the acquisition of new biochemical functions is the ability of a ribozyme to accommodate diffusible substrates. We are analyzing the mechanism and catalytic requirements of kinase ribozymes. RNA-catlyzed phosphorylations are attractive to study for several reasons. Among them, phosphoryl transfer is one of the most important and ubiquitous reactions in any type of metabolism, and of fundamental biological and evolutionary significance. The present work describes a kinase ribozyme, MK28-2, which was selected to use GTP(γS) as (thio)phosphoryl donor. MK28-2 promotes (thio)phosphorylation of two distinct 2’ hydroxyls that are widely separated in primary sequence. The secondary structure of the active molecule was obtained by enzymatic probing and nucleotide requirements for catalysis and tertiary interactions identified by mutational analysis. The path and fate of double (thio)phosphorylation was followed with the aim of understanding the catalytic potential of the active site. Metal requirements for optimal activity were also investigated, showing a high dependence of the ribozyme on magnesium and copper ions. Moreover, this RNA was shown to be the first kinase ribozyme which activity is influenced by pH variations. The results obtained with these experiments provide new prospects on the mechanism of catalysis and substrate binding demand for kinase ribozymes. These studies will help delineate the catalytic potential of metabolic ribozymes in the contexts of both an RNA World and an RNA-based engineered metabolism.

6

Positive regulation of RpoS by small RNAs and the role of Hfq Toby Soper1, Pierre Mandin2, Nadim Majdalani2, Susan Gottesman2, and Sarah A. Woodson1,3,* 1

) Johns Hopkins University, T. C. Jenkins Dept. of Biophysics, 3400 North Charles Street, Baltimore, MD 21218. 2) Laboratory of Molecular Biology, Bldg. 37, Rm. 5132, National Cancer Institute, Bethesda, Maryland, 20892. *Corresponding author. (410) 516-7348, Fax: (410) 516-4118, [email protected] Bacterial small non-coding RNAs (sRNAs) carry out both positive and negative regulation of gene expression by pairing with mRNAs. This regulation often requires the abundant Sm-like RNA chaperone protein Hfq. Three sRNAs, DsrA, RprA, and ArcZ, positively regulate translation of the stationary phase sigma factor RpoS, each pairing with the 5’ leader to open up an inhibitory stem-loop that prevents ribosome binding. We previously showed that an (AAN)4 motif in the rpoS leader upstream of the inhibitory stem is required for the RNA chaperone activity of Hfq. As measured in vitro using native PAGE, rpoS leaders long enough to contain the (AAN)4 repeat bind Hfq tightly and form ternary complexes with DsrA, RprA, and ArcZ. Hfq strongly stabilized complexes of the long rpoS leader with DsrA at 25ºC, and RprA and ArcZ at 37ºC. We show that the stabilities of the complexes between the sRNAs and the rpoS leader correlated well with ability of the sRNAs to activate rpoS::lacZ fusions in vivo. We also demonstrate the importance of the (AAN)4 repeat motif for sRNA activity in vivo. Hfq has separate binding sites for A-rich and U-rich RNA on opposite sides of its homo-hexameric ring structure. However, we show that Hfq cobinding to the two RNAs is insufficient to promote DsrA annealing to the rpoS leader and that stable ternary complexes require RNA•RNA pairing. Rather, Hfq acts by restructuring the rpoS leader to facilitate DsrA pairing.

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SOMAmers: Proteomics to lung cancer to a Wellness Chip Larry Gold SomaLogic, Boulder, Colorado, USA We figured out a way to make novel aptamers, called SOMAmers. The SOMAmers, made of modified single-stranded DNA, have high affinities for their target human proteins, and in addition have very slow off-rates. The high binding quality of these special aptamers is supported by a crystal structure of one such SOMAmer with its protein partner. We also figured out a way to use the SOMAmers to do deep proteomics. Today we measure 1,100 human proteins in serum, plasma, or tissue extracts, using only 15 µl of sample. The measurements are accurate (CVs of ~ 5%) and very sensitive; more than half the proteins are measured with limits of detection below pM. The combination of reagents and a novel assay has made biomarker discovery relatively easy. And thats what we do ... We get samples from healthy and sick people and do an unbiased experiment by asking if any proteins on the menu go up or down in the blood of sick people. We have found many blood biomarkers for lung cancer, mesothelioma, pancreatic cancer, and more. Lung cancer is the leading cause of cancer deaths. Most cases are diagnosed at an advanced stage. Patients diagnosed at an early stage who have surgery experience an 86% overall 5-year survival thus one needs earlier detection of lung cancer. I will share data from a large clinical trial (almost 1,400 subjects) from four independent studies of long-term tobacco-exposed populations. We identified 44 candidate biomarkers, from which a 12- protein panel was constructed. The panel identified NSCLC from controls with 91% sensitivity and 84% specificity in the training set, and 89% sensitivity and 83% specificity in the blinded, independent verification set. Performance was similar for early and late stage disease. We also have compared protein concentrations in NSCLC biopsy materials with literature values for mRNA concentrations in similar biopsies. The correlations between proteins and their mRNAs are poor, suggesting that a discovery proteomics platform might be essential for understanding NSCLC biology. Finally, with only some trepidation, I will describe our goal of transforming medicine through a Wellness Chip, aimed at the most devastating diseases for which early diagnosis and action could be life-saving.

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Mapping the competing barriers that define alternative RNA folding pathways 1

2

2

1

Jörg Schlatterer , Joshua Martin , Alain Laederach , Michael Brenowitz 1

2

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461

Computational and Structural Biology Department, Wadsworth Center, Albany, NY 12208

The folding of large RNA molecules into biologically active structures often traverses rugged energy landscapes populated by intermediates possessing either native or non-native structure. To date, a comprehensive and quantitative analysis of the barriers that define the folding landscape has remained experimentally difficult to attain. By combining high-throughput chemical probing and kinetic modeling we report the activation energies for the seven forward 2+

transitions that are resolved for the Mg -mediated time dependent folding of the Tetrahymena ribozyme. We explore the partitioning of activation barriers into their enthalpic and entropic components for individual steps of folding reactions which include transitions between the unfolded RNA ensemble (U), the intermediates I1 (only the P4-P6 ‘scaffold’ domain is formed) and I2 (both the peripheral helices and the P4-P6 domain are formed), and the folded RNA (F).

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Time-Resolved SAXS Reconstructions Reveal a Kinetic Intermediate in RNA Folding Steve Meisburger1, Suzette Pabit1, Li Li1, Joshua Blose1, Krista Brooks2, Ken Hampel2, and Lois Pollack1 1

School of Applied and Engineering Physics, Cornell University; Department of Microbiology and Molecular Genetics, University of Vermont Presenting author: [email protected], (607)-254-6599 FAX (607)-255-7658

2

Using complementary time-resolved biochemical and x-ray probes of RNA structure in solution, we study the cation-induced folding of the glmS ribozyme, a metabolite-sensing RNA switch that regulates gene expression in bacteria. Hydroxyl radical footprinting has shown that tertiary contacts form during a concerted folding step. From small angle x-ray scattering experiments performed at CHESS, we find that tertiary contact formation is preceded by the collapse of the molecule to a relatively compact intermediate state. The transition to a final state consistent with the crystal structure correlates temporally with changes in hydroxyl radical protection. We discuss a method for using ab initio reconstructions to obtain average geometrical parameters for the intermediate state and quantify the spatial extent of the molecule as it folds.

10

Alternative assembly pathways of ribosomal 30S subunits in thermophilic bacteria Nathan Napper and Gloria M. Culver University of Rochester Rochester, NY 14627 (585) 276-3602, [email protected] The ribosome is a large macromolecular complex composed of two asymmetric subunits, each performing critical roles during the translation process. The form and function of this essential cellular machinery has been actively studied, both in vivo and in vitro for several decades. Studies of bacterial 30S subunit assembly have predominantly used E. coli as the model organism. This work has contributed greatly to the knowledge of how 16S rRNA and 20 r-proteins combine to form a functional 30S subunit. It remains unclear, however, if all bacteria use an assembly pathway similar to that of E. coli, or if alternative pathways are used. Bacteria are capable of surviving in diverse and extreme ecological environments, such as extreme temperatures. Due to the temperature dependent nature of 30S subunit assembly in vitro, the way in which thermophilic bacteria assemble ribosomal 30S subunits is of particular interest. To this end we have studied ribosomal 30S subunit assembly in two bacterial species with an optimum growth temperature of 55°C, Geobacillus stearothermophilus and Geobacillus kaustophilus. Using sucrose gradient sedimentation, tRNA binding, mass spectrometry and 2D gel electrophoresis we have identified and characterized two distinct assembly intermediate populations present in the ribosomal 30S subunit assembly pathway of these bacterial species. These intermediate populations differ in sedimentation properties, function and r-protein composition. This alternative pathway may shed light, both onto the flexibility of assembly of the small ribosomal subunit as well as present novel targets for innovative antibacterial compounds.

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Nucleotides that are essential but not conserved Rob Knight Associate Professor, University of Colorado at Boulder Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

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Automated RNA Tertiary Structure Prediction Matthew G. Seetin and David H. Mathews Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester Medical Center, 601 Elmwood Ave, Box 712, Rochester, NY 14624 Correspondence should be addressed to Matthew G. Seetin, Phone: (585) 2764337. Fax: (585) 275-6007. E-mail: [email protected] A novel protocol employing simulated annealing and steered molecular dynamics for all-atom RNA tertiary structure prediction is presented. The restraints are derived from secondary structure, coaxial stacking predictions for helices in junctions, co-variation analysis, and, when available, cross-linking data. The protocol is tested on the Alu domain of the mammalian signal recognition particle RNA, the tRNAPhe of Saccharomyces cerevisiae, the hammerhead ribozyme, and the hepatitis C virus internal ribosomal entry site. The resulting pool of decoy structures is scored with a scoring function that maximizes the radius of gyration and number of base-base contacts to identify the best predicted structure. These predictions are compared with the crystal structures using both root mean square deviation (RMSD) and the accuracy of base-base contacts. This simple ab initio approach using the above restraints is sufficient to make accurate predictions of the structures, with, for example, an RMSD of 5.0 Angstroms for all heavy atoms in the mammalian SRP Alu domain.

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RNAmutants: A computational framework to explore the mutational landscape of structural RNAs. Theory and applications. Jérôme Waldispuhl School of Computer Science & McGill Centre for Bioinformatics, McGill University, 3480 University Street, Montreal, Quebec H3A 2A7, Canada Phone: +1-514-398-5018, Fax: +1-514-398-3883, Email: [email protected] The understanding of the relationship between RNA sequences and structures and the quantification of the structural changes produced by point-wise mutations is essential to decipher evolutionary processes but also to predict deleterious mutations to support mutagenesis experiments, analyze and eventually fix disease-related mutations, and design novel RNA molecules. We designed the first algorithm, RNAmutants, for exploring in polynomial time and space complete RNA sequence-structures maps [1]. Thus opening the door to large-scale studies. Through this efficient exploration of the mutation landscape, we apply statistical mechanics techniques for estimating the thermodynamical pressure that has been applied on sequences. We have successfully applied RNAmutants to investigate deleterious mutations (mutations that radically modify secondary structure) in the Hepatitis C virus cis-acting replication element and to evaluate the evolutionary pressure applied on different regions of the HIV transactivation response element. We also provided evidence that the 3’ UTR of the GB RNA virus C has been optimized to preserve evolutionarily conserved stem regions from a deleterious effect of pointwise mutations. More recently, we applied our algorithms to develop a novel methodology for designing RNA sequences with predetermined secondary structures. The later produces artificial sequences with striking similarities to “natural” RNAs found in the Rfam database. Joint work with: Bonnie Berger, Peter Clote, Srinivas Devadas, Rafik Draoui, Alex Levin, Mieszko Lis, Charles W. O’Donnell and Yann Ponty.

[1] Jérôme Waldispuhl, Srinivas Devadas, Bonnie Berger and Peter Clote, Efficient Algorithms for Probing the RNA Mutation Landscape, PLoS Comp. Bio., 2008. 14

Higher-order structure of RNA by MS-based approaches Dan Fabris The RNA Institute, University at Albany The observation that less than 1.5% of the human genome codes for actual proteins has lead to the realization that sequence information alone is insufficient to elucidate the function of the vast majority of nucleic acids in living organisms. The recent discovery of riboswitches has keenly reasserted the critical role played by higher-order structure in determining the function of non-coding elements. Beyond sequencing, approaches based on mass spectrometry (MS) can provide direct information about base-pairing and long-range interactions, which respectively define the secondary and tertiary structure of nucleic acids. The ability to observe intact assemblies with other nucleic acid elements and cognate proteins enables the investigation of their quaternary structure. For these reasons, we have been employing electrospray ionization (ESI) with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to investigate the structure-function relationships of the 5' untranslated region (5'UTR) of the genome of HIV-1 and its assemblies with the chaperone nucleocapsid (NC) protein. The presentation will illustrate the development of solution and gas-phase approaches for elucidating the 3D structure and spatial organization of RNA and its functional assemblies with proteins and small molecule ligands.

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The tertiary structure and folding pathways of group II introns: paradigms for the assembly of large, multidomain RNAs and RNPs Anna Marie Pyle Department of Molecular, Cellular and Developmental Biology and Department of Chemistry, Yale University Group II introns are large ribozymes that catalyze their own self-splicing from precursor RNAs. The free introns can catalyze integration into duplex DNA through reverse-splicing reactions that can lead to intron mobility and retrotransposition within and among genomes. Group II introns therefore represent an important class of mobile genetic element that has played an important role in the evolution and diversification of terrestrial genomes. Based on their mechanistic and structural parallels with spliceosomal RNAs, they may also have played in important role in the evolution of eukaryotic RNA processing systems. In our laboratory, we study the tertiary structure and folding pathways of group II introns. We have found that group II introns can fold directly and faithfully to the native state through a series of obligate intermediates. Although group II introns can fold autonomously, certain proteins play a key stimulatory role during the various stages of folding and assembly in-vivo. We recently solved the crystal structure of a group II intron, which has revealed networks of tertiary interactions and scaffolding that are critical for folding, stability and catalysis. By examining the structure and kinetic properties of the folded intron, and of intermediates along the assembly pathway, we hope to define the driving forces for folding of large RNAs, and to describe the various ways that proteins facilitate this process.

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POSTER SESSION I 4:30 pm – 5:30 pm Friday, Oct 8, 2010

Development of the sxRNA platform; A trans-acting RNA switch Arthur Beauregard, Sabari Jayaseelan, Francis Doyle and Scott Tenenbaum College of Nanoscale Science and Engineering. Nanobioscience Constellation, University at Albany-SUNY, Albany, New York 12203, USA We currently are developing a technology termed structurally-interacting RNA (sxRNA). The approach consists of using an engineered “bait-RNA,” a “triggerRNA” that exists in the cell or other organism, and an RNA-binding protein (RBP) that binds to the bait when activated by the trigger. The engineered bait has been altered so it does not form a proper structure for RBP binding but will undergo a conformational change in the presence of the trigger-RNA, which induces RBP association and enhances translation of an upstream gene. This approach could be used to turn-on biochemical reactions and other targeted cellular processes. We have identified several naturally accruing bait and triggerRNAs and have used this information to design custom sxRNAs using a bioinformatic approach. We are currently studying the in vitro translation of the upstream gene following the conformational change of the bait in the presence of the trigger and the association of the RBP. Studies of the sxRNA platform will be moved to an in vivo system following in vitro optimization. The conformational change of the sxRNA platform can take many forms, but typically activates so as to suppress, or modulate a cascade of biochemical events, leading a therapeutic effect. The sxRNA platform could also be used to target a viral RNA with the result that all virus-infected cells die. The potential for the sxRNA platform is universal, in that it can be utilized in any situation in which a unique trigger RNA is expressed, which can take the form or many types of RNA.

P-1

ProbKnot: Fast Prediction of RNA Secondary Structure Including Pseudoknots Stanislav Bellaousov and Dave Mathews* Department of Biochemistry & Biophysics, University of Rochester *To Whom Correspondence should be addressed: Tel: 1-585-276-4338. Fax: 1585-275-6007. E-mail: [email protected] It is a significant challenge to predict RNA secondary structures including pseudoknots. Here, a new algorithm capable of predicting pseudoknots of any topology, ProbKnot, is reported. ProbKnot assembles maximum expected accuracy structures from computed base pairing probabilities in O(N2) time, where N is the length of the sequence. The performance of ProbKnot was measured by comparing predicted structures to known structures for a large database of RNA sequences with fewer than 700 nucleotides. The percent of known pairs correctly predicted was 69.3%. Additionally, the percentage of predicted pairs in the known structure was 61.3%. This performance is the highest of four tested algorithms that are capable of pseudoknot prediction. The program is available for download at: http://rna.urmc.rochester.edu/RNAstructure.html

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Molecular Determinants For hSLIP1 Recognition By Factors That Modulate mRNA Processing And Stability Aishwarya Bhaskar, Nitin Bansal, Patrick Itotia, Ming Jing Wu, Roopa Thapar Hauptman-Woodward Medical Research Institute and Department of Structural And Computational Biology, SUNY, Buffalo, NY 14203 Tel: 716-898-8687, Fax: 716-898-8600, E:mail: [email protected] The mammalian protein SLIP1 is a HEAT repeat containing protein that is structurally related to the middle domain of eIF4G (MIF4GD). It has been implicated to play a role in translation initiation of histone mRNAs via its interaction with the histone mRNA-specific factor SLBP. To identify candidate effector proteins that interact with SLIP1 to modulate gene expression, we screened a mammalian yeast two-hybrid library using hSLIP1 as bait. Functionally relevant interactions with these effectors were confirmed by coimmunoprecipitation with hSLIP1 in Hela cells. Proteins that we have established as SLIP1 targets include SLBP, c-Myc, hERI1/3’hExo, hDbp5, two subunits of eIF3, and a subunit of the Lsm complex. To identify key residues on hSLIP1 that impart specificity towards these interactors we used the crystal structure of the zebra fish orthologue of SLIP1 (PDB code 2I2O) along with evolutionary conservation profiles of amino acids in SLIP1. Hot spots for binding of SLIP1 interactors were identified using alanine scanning mutagenesis and Surface Plasmon Resonance (SPR). hSLIP1 mutants that impair specific functional pathways by abolishing interaction with these effector proteins are being characterized in vivo. Ongoing studies in the laboratory to understand the functional and structural roles of SLIP1-effector interactions in regulating RNA processing and turnover will be presented.

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Effects of protecting osmolytes on electrostatic interactions among DNA duplexes Joshua M. Blose, Suzette A. Pabit, Steve P. Meisburger, Li Li, Christopher D. Jones, and Lois Pollack* School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853 *Phone: (607) 255-8695, Email: [email protected] Osmolytes are small, chemically diverse, intracellular organic solutes that function as a vital component of cellular stress response. Protecting osmolytes enhance protein stability via preferential exclusion, where denaturation of the protein in the presence of the osmolyte is less favorable than in an aqueous environment. Thus, the correct ratios of protecting to non-protecting osmolytes and protecting osmolytes to ions are critical to maintain protein structure or protein-nucleic acid interactions. In contrast to osmolyte effects on protein stability, structure, and function, there is much less understood concerning the effects of osmolytes on nucleic acids. Although non-protecting osmolytes can destabilize both protein and nucleic acid structures, protecting osmolytes have different effects on nucleic acid structure depending on polymer type and structural complexity, and the general the effects of osmolytes on the ion atmosphere surrounding nucleic acids is not well understood. Thus, in order to begin probing the effects of osmolytes on nucleic acid electrostatics and the ion atmosphere, we examined the structure of a 25-bp DNA duplex using small angle x-ray scattering (SAXS) techniques in the presence and absence of sucrose, a protecting osmolyte and important contrast matching agent in SAXS studies of protein-nucleic acid complexes. Results will be discussed.

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Interdependencies govern multi-domain architecture in E.coli small subunit assembly Deepika Calidas & Gloria M. Culver* Department Of Biology, University of Rochester, Hutchison Hall 301, Rochester, NY 14627 *Corresponding Author, Phone: 585-276-3602, Fax: 585-275-2070, E-mail: [email protected] The 30S subunit is composed of four structural domains, the body, platform, head and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe (II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multi-domain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in an appropriate architecture in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, the appropriate architecture of the critical elements of the 5' domain is established only upon assembly of the body. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

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Anticodon loop modifications modulate structural flexibility in E. coli tRNAArg1,2 that lacks the U-turn conformation in solution William A. Cantara*1,2, Yann Bilbille1, Jia Kim1 & Paul F. Agris2 1

Department of Molecular & Structural Biochemistry, North Carolina State University, Raleigh, NC 27695 2 Institute for RNA Science & Technology, University at Albany, Albany, NY 12222 Telephone: (518) 437-4448; Fax: (518) 437-4456; Email: [email protected] Of the six codons that are decoded by tRNAArg in E. coli, three are read by the isoacceptors tRNAArg1, 2. These isoacceptors differ only in the identity of the residue at position 32 in the anticodon stem and loop domain (ASL) as either cytosine or 2-thiocytosine (s2C32) for tRNAArg1 and tRNAArg2 respectively. These isoacceptors also contain modifications at positions 34 (inosine, I) and 37 (2methyladenosine, m2A37). To investigate the roles of these modifications in proper folding of the ASL, six ASL constructs differing in their array of modifications were analyzed by biophysical spectroscopic methods as well as functional binding assays. Thermal denaturation and circular dichroism spectroscopy showed that the modifications contribute competing thermodynamic and base stacking properties. While spectroscopic methods indicate that ASL modifications contribute significant differences in structural properties, native gel electrophoresis and NMR spectroscopy clearly show that the equilibrium solution conformations of the ASLs are nearly identical, but do not possess the invariant U-turn structure needed for binding to the ribosomal A-site. Of the six ASL variants, all are able to bind to the ribosome in the presence of the cognate CGU codon and the three constructs containing I34 bound to CGC, while both s2C32 and m2A37 restrict binding to CGA. Taken together, the results suggest that chemical modifications modulate the flexibility of the loop, allowing induced conformations on the ribosome that can restrict binding to specific rare codons.

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Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry Venkata R. Chalamcharla, Joan Curcio and Marlene Belfort Group II introns are self-splicing RNAs found in eubacteria, archaea and eukaryotic organelles. They are mechanistically similar to the metazoan nuclear spliceosomal introns; therefore, group II introns have been invoked as the progenitors of the eukaryotic pre-mRNA introns. However, the ability of group II introns to function outside of the bacterial-derived organelles is debatable, since they are not found in the nuclear genomes of eukaryotes. Here, we show that the Lactococcus lactis Ll.LtrB group II intron splices accurately and efficiently from different pre-mRNAs in a eukaryote, Saccharomyces cerevisiae. However, a premRNA harboring a group II intron is spliced predominantly in the cytoplasm, is subject to nonsense-mediated mRNA-decay (NMD), and the mature mRNA from which the group II intron is spliced is poorly translated; the mechanism of translational repression is currently under investigation. In contrast to a group II introncontaining transcript, a pre-mRNA bearing the Tetrahymena group I intron or the yeast spliceosomal ACT1 intron at the same location is not subject to NMD, and the mature mRNA is efficiently translated. Thus, a group II intron can splice from a nuclear transcript, but RNA instability and translation defects would have favored intron loss, or evolution into protein-dependent spliceosomal introns, consistent with the bacterial group II intron-ancestry hypothesis.

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Atomistic Simulations of the Force-Induced Dissociation of Retroviral RNA Kissing-Loops Alan A. Chen and Angel García* Rensselaer Polytechnic Institute 1C25 JR Science Center 110 8th Street, Troy, NY 12180 * [email protected], (518)-276-4206, fax: (518)-276-2955 Retroviruses require two copies of their ssRNA genomes in order to form infectious virus particles. This is accomplished via a Dimerization Initiation Site (DIS), which forms a rivet-like “kissing-loop” that binds the two genomes together. Retroviral DIS kissing loops have been shown to be unusually resistant to heat denaturation or mechanical pulling, given the small number (2-6) of Watson-Crick base-pairs involved. This high mechanical stability is crucial for retroviral fitness, as mutations that destabilize the DIS loop in-vitro also result in greatly reduced virus infectivity rates in-vivo. Therefore, understanding the physical determinants that give rise to enhanced DIS kissing-loop stability may prove to be a successful route to designing new anti-retroviral therapeutics. The Moloney Murine Leukemia Virus (MMLV) serves as a particularly tractable model system due to its small size, as it comprised of two GACG tetraloops joined by only two loop-loop base-pairs. Our collaborator, Pan Li at SUNY Albany, has shown, using single-molecule pulling experiments, that it requires more force to break the MMLV DIS than would be required to unfold an entire 11-bp hairpin. Using a combination of equilibrium and non-equilibrium all-atom molecular dynamics simulations, we have developed a detailed model for the kinetic intermediates of the force-induced dissociation of the MMLV DIS kissing-loop. We find that transient stacking interactions by the adjacent, unpaired loop adenines are able to greatly enhance the lifetimes of the two loop-loop base-pairs, and that the breaking of these stacking interactions are the rate-limiting step for force-induced dissociation of the MMLV DIS complex.

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Topological frustration in the folding intermediates of the Twort and Azoarcus group I introns Chunxia Chen1,2†, Somdeb Mitra5, Magdalena A. Jonikas3,4, Michael Brenowitz5, and Alain Laederach1,2§ 1

Department of Biomedical Sciences, University at Albany, Albany, NY 12208 Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, NY 12208 3 Harvard Medical School, Boston, MA 02115 4 Division of Emergency Medicine, Children’s Hospital Boston, Boston, MA, 02115 5 Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY- 10461 2

§

To whom correspondence should be addressed. Phone: (518) 486 4103, fax: (518) 474 3181, e-mail [email protected]

Topological frustration is a contributing factor to the lifetime of RNA folding intermediates, requiring significant unfolding and subsequent refolding to allow formation of the active conformation. We investigate the role of RNA topology in folding kinetics by comparing intermediates modeled from time-resolved hydroxyl radical (•OH) fooptrinting data collected on the Twort and Azoarcus group I introns. Kinetic analysis of the data reveals an inverted tertiary contact formation order for these two structurally and phylogenetically related RNAs. We modeled the kinetic folding intermediates with the Nucleic Acid Simulation Tool (NAST), which uses a knowledge-based coarse-grained representation of RNA to efficiently sample topologies given a set of experimental (in this case •OH footprinting) constraints. We investigate the role of the unfolded conformation on topological frustration by starting our folding simulations from a diverse set of unfolded structures. Interestingly, the major determining factor in whether the introns become trapped in a topologically frustrated state is the initial conformation, irrespective of the folding pathway taken. Our results therefore add to the growing body of evidence pointing to the initial conformation as a major determinant of RNA folding kinetics. Additionally, our work suggests that a nonrandom sampling of the unfolded conformations explains the observed differences in relative flux through RNA folding pathways.

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A protective partnership between Group II intron RNPs and host ribosomes L. Contreras-Martin, T.Huang, C.L.Piazza, J. Danserau M. Belfort Group II introns are mobile retroelements, found in archaea, bacteria, and the organelles of eukaryotes. They insert site-specifically into an intron-less DNA by passing through an RNA intermediate. During group II intron mobility, a highly stable complex forms between the intron RNA (LtrB) and the intron encoded protein (LtrA); this RNP plays a crucial role in facilitating the targeting and invasion of the DNA substrate. Although several in vitro studies have focused on the types of specific RNA-protein interactions that lead to the formation of this ribonucleoprotein complex, complete understanding of how this RNP forms intracellularly and remains stable is unclear. Moreover, little is known about how the formation of this complex in vivo relates to the activity of the group II intron. Due to the natural presence of an open reading frame (ORF) within an essential domain (dIV) of the intron, it is intriguing to consider how the recruitment of ribosomes could affect the stability and function of the intron in vivo. We have developed a purification protocol for the isolation of native, active, group II intron RNPs from Lactoccocus lactis that have allowed us to confirm a strong interaction between our group II intron RNP and host ribosomes. We have made significant progress in characterizing the extend to which these two RNPs associate and the biological implications of this interaction.

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The Impact of Cis-acting Polyadenylation Elements on Alternative Polyadenylation Sarah K. Darmon, Ashley L. Cornett, Bin Tian and Carol S. Lutz* Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School and the Graduate School of Biomedical Sciences, Newark, NJ 07103 *Corresponding Author: 973-972-0899 phone, 973-972-5594 fax, [email protected] email Polyadenylation is an mRNA 3’ end processing event that contributes to the regulation of gene expression by affecting turnover, stability, export, and translation into protein. More than half of all mammalian mRNAs are now known to have more than one polyadenylation signal, resulting in a large number of mRNAs that are alternatively polyadenylated. We wanted to explore cis-acting RNA elements that contribute to usage and regulation of these alternative polyadenylation signals. We used bioinformatics studies which identified specific RNA sequence motifs as potential auxiliary elements both upstream and downstream of the core polyadenylation signals. We used this predicted information to direct in vivo validation studies where a battery of auxiliary elements were placed either upstream or downstream of the polyadenylation signal in the 3’ UTR of luciferase reporter constructs. Addition of upstream auxiliary polyadenylation elements enhanced reporter expression three- to four-fold, while addition of auxiliary downstream polyadenylation elements gave approximately a two-fold enhancement. We have also examined the impact of RNA secondary structure on polyadenylation as well as interactions of miRNAs with 3’UTRs.

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A sequence similar to tRNA 3 Lys gene is embedded in HIV-1 U3R and promotes minus-strand transfer Laura DiChiacchio, Dorota Piekna-Przybylska., Robert Bambara, David Mathews Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. We discovered a sequence in the U3 region of HIV-1 that hybridizes with tRNAlys3 with significantly high stability. This region includes a 9-nt sequence that has been previously shown to improve minus-strand transfer efficiency through interactions with tRNAlys3. The U3 region is highly complementary to tRNAlys3, excluding a 29-nt segment present where a tRNA intron would be expected. This structure suggests that a host tRNA gene was incorporated into the HIV-1 genome at some point in evolution and was maintained by selective pressure. In vitro experiments show that this U3 segment improves minus-strand transfer efficiency beyond that of the 9-nt sequence alone, suggesting that the role of this sequence in HIV-1 replication is the mechanism driving its conservation over time.

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Multiple small RNAs identified in Mycobacterium bovis BCG, M. tuberculosis, and M. smegmatis Jeanne M. DiChiara1, Lydia M. Contreras-Martinez1, Jonathan Livny2,3, Dorie Smith1, Kathleen A. McDonough4, and Marlene Belfort1,4 1

Division of Genetics and 4Division of Infectious Diseases, Wadsworth Center, Albany, NY 12208 2 Broad Institute of MIT and Harvard, Cambridge, MA 02142 3 Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

Tuberculosis (TB) is a major global health problem, infecting millions of people each year. The causative agent of TB, Mycobacterium tuberculosis (Mtb), is one of the world’s most ancient and successful pathogens. Until recently, no work on small regulatory RNAs had been performed in Mtb. Regulatory RNAs are found in all three domains of life, and have already been shown to regulate virulence in well-known pathogens. This work describes the discovery of 35 novel small RNAs (sRNAs) in the TB-complex M. bovis BCG. We achieved this using a combination of experimental and computational approaches. Putative homologues of many of these sRNAs were identified in M. tuberculosis and/or M. smegmatis. The sRNAs that are expressed in both BCG and the non-pathogenic M. smegmatis could be functioning to regulate conserved cellular functions. In contrast, those sRNAs identified specifically in M. tuberculosis could be functioning to regulate virulence, thus rendering them potential targets for novel antimycobacterials. Of particular interest to us are the sRNAs Mcr9, Mcr11, and Mcr13. Mcr9 is upstream of the ilvB1 gene, which has been shown to be essential for virulence in mice. Mcr11 is located between the genes Rv1264, an adenylyl cyclase, and Rv1265, a cAMP-induced gene. Due to its location, this sRNA could regulate Rv1265, and may therefore be involved in cAMP metabolism. Mcr13 is antisense to two genes in the ESX-1 locus, espG1 and espH. This sRNA could regulate one or both of these genes and is therefore of great interest and is currently being studied further.

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Widespread Antisense Transcription in Escherichia coli James Dornenburg1, Anne Stringer1, Christopher Karch2 and Joseph Wade1,2,3 1

Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY, 12208 2 Department of Biomedical Sciences, School of Public Health, University at Albany, SUNY, Rensselaer, NY, 12144 3

[email protected]

Tel: (518) 474 5727

Fax: (518) 474 3181

Of the ~4,000 genes described for the model bacterium, Escherichia coli, only 8 overlap another gene by more than a few base pairs. Using a next generation sequencing approach to identify RNA 5’ ends, we discovered >1,000 novel RNAs that are encoded antisense to protein-coding genes. We validated these RNAs using bioinformatic and experimental approaches. These antisense RNAs are likely to be non-coding and we present evidence that they play an important regulatory role. Our data also suggest the existence of >1,000 novel RNAs that initiate within protein-coding genes in the sense orientation. We propose that the novel sense and antisense transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene.

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Application of Secondary Structure Prediction Tools to Find Potential trans-acting RNA Stabilizers of RBP Binding Motifs Francis Doyle College of Nanoscale Science and Engineering. Nanobioscience Constellation, University at Albany-SUNY, Albany, New York 12203, USA RNA-binding proteins (RBPs) perform many roles in posttranscriptional gene regulation. RBPs often bind to a particular "stem-loop" type secondary structure motif found in a subset of associated mRNAs, such as the histone stem loop found at the 3' end of metazoan histone messages. A common element found (in cis) in various functional RNA structures is the three-way junction, which occurs when three RNA helices meet. We have previously hypothesized that such elements may occur in trans and provide a positive bias toward RBP recognition motif formation. Bioinformatic tools for predicting RNA secondary structures (both in cis and in trans) through analysis of energy states may be applied to perform a high throughput search for mRNA/ncRNA pairings with a potential for this type of interaction.

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The Role of Sm-like Protein Hfq in RNA Annealing Kevin Doxzen, Toby Soper, Sarah A. Woodson* Johns Hopkins University, T. C. Jenkins Dept. of Biophysics, 3400 North Charles Street, Baltimore, MD 21218. *Corresponding author. (410) 516-7348, Fax: (410) 516-4118, [email protected] Small RNAs (sRNAs) regulate bacterial response and adaptation to environmental stress. This RNA regulation mechanism requires the Sm-like binding protein, Hfq, which promotes annealing of complimentary RNAs. The distal face of Hfq interacts with poly(A) sequences, while the proximal face interacts with poly(U) sequences. The sRNA DsrA binds to the rpoS mRNA, opening an inhibitory stem loop in the mRNA, and consequently initiating translation of the RpoS sigma stress response factor in Escherichia coli. Hfq binding to an (AAN)4 repeat element upstream of the inhibitor stem loop on rpoS mRNA has been shown to be required to promote DsrA annealing. To further understand the role of Hfq in RNA annealing, we studied the formation of the ternary complex between Hfq, sRNA DsrA, and mRNA rpoS mRNA. Two mutations were chosen to disrupt RNA binding to either the proximal or distal face of the Hfq hexamer. Fluorescence Anisotropy experiments were used to study the binding affinity of Hfq mutants to poly(U) oligomers. Y25D, on the distal face of Hfq, showed only a two fold decrease in binding affinity; whereas K56A, on the proximal face, showed a 5-fold decrease. Native gel mobility shift assays showed that only the K56A mutant formed a ternary complex with DsrA and rpoS RNAs. In contrast, the Y25D mutation inhibits rpoS mRNA binding and also fails to form a ternary complex. These results support previous results showing that Hfq binding to A-rich sequences in rpoS mRNA is critical for rpoS regulation.

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Self-processing of the R2 retrotransposon from a 28S rRNA cotranscript Danna G. Eickbush and Thomas H. Eickbush Department of Biology, University of Rochester, Rochester, NY 14627 Phone:585-275-7247, Fax:585-275-4070, [email protected] The non-long terminal repeat (non-LTR) retrotransposon R2 inserts into the 28S rRNA genes of many animals. Expression of the element appears to be by cotranscription with the rRNA gene unit. Processing of the rRNA co-transcript at the 5' end of the R2 element in Drosophila simulans is rapid and utilizes an unexpected mechanism. Using RNA synthesized in vitro, the 5' untranslated region of R2 was shown capable of rapid and efficient self-cleavage of the 28S– R2 co-transcript. The 5' end generated in vitro by the R2 ribozyme was at the identical position found for in vivo R2 transcripts. The RNA segment corresponding to the R2 ribozyme could be folded into a double pseudoknot structure similar to that of the hepatitis delta virus (HDV) ribozyme. Remarkably, 21 of the nucleotide positions in and around the active site of the HDV ribozyme were identical in R2. R2 elements from other Drosophila species were also shown to encode HDV-like ribozymes capable of self-cleavage. Tracing their sequence evolution in the Drosophila lineage suggests that the extensive similarity of the R2 ribozyme from D. simulans with that of HDV was a result of convergent evolution not common descent. These findings provide an explanation for the sequence variation at the 5' end of R2 elements and how second strand DNA synthesis may be initiated during retrotransposition.

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mRNA Translocation Occurs During the Second Step of Ribosomal Intersubunit Rotation Dmitri N. Ermolenko1,2 and Harry F. Noller2 1

Department of Biochemistry and Biophysics and Center for RNA Biology: From Genome to Medicine, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642 2 Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064 Correspondence should be addressed to Dmitri Ermolenko, 585-275-3704 (phone), 585-271-2683 (fax), [email protected] (email) During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor EF-G. Based on cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We have used a combination of two fluorescencebased approaches to study the timing of these events: Intersubunit FRET measurements to observe relative rotational movement of the subunits and a fluorescence quenching assay to monitor translocation of mRNA. Under conditions where translocation is slowed, two rotational events are resolved. Binding of EF-G·GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. Comparison of the rates of these movements reveals that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the transition from the hybrid state to the classical state.

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SHAPE experiments on the human telomerase RNA pseudoknot subdomain Alexis Catherine Espinosa State University of New York at Albany,1400 Washington Ave., Albany, NY Dr. Carla Theimer Phone:518.591.8875, fax:518.442.3462 and e-mail address:[email protected] Telomerase is an essential ribonucleoprotein (RNP) complex, which is responsible for the formation and maintenance of telomeres which preserve the integrity of genetic information during normal DNA replication. Human telomerase is composed of two catalytically essential constituents, a 451 ntd RNA, the telomerase RNA (hTR), and a 120 kDa protein, the telomerase reverse transcriptase (hTERT); as well as a variety of accessory proteins. Mutations in both hTR and hTERT, have been linked to diseases of the hematopoietic system. hTR contains the pseudoknot and CR4-CR5 domains, which are required for binding hTERT and telomerase catalysis, as well as the H/ACA-scaRNA domains, which are required for 3’ end maturation and localization in vivo. Of the 33 disease mutations identified in hTR, 21 of them are found in the pseudoknot domain. The biological importance of this region has lead to a number of structural investigations using NMR spectroscopy; where the importance of a hairpin- pseudoknot conformational equilibrium has been proposed and structural features have been determined for shortened constructs at high resolution. Here, we further investigate this proposed conformational equilibrium using SHAPE technology to examine the entire pseudoknot domain of wild type hTR. In addition to the wild type hTR RNA, two mutations GC(107,108)AG and 177ΔU, which have been shown to directly disrupt the conformational equilibrium will also be investigated. Further investigation will be necessary in order to characterize conformational equilibrium involvement in hTR- hTERT binding interactions.

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POSTER WITHDRAWN

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Effect of Single Nucleotide Polymorphism (SNP) in the 5’ UTR in SERPINA1 on the secondary structure of RNA Chetna Gopinath1, 2, Lauren Davis-Neulander2, Matt Halvorsen1, 2, Alain Laederach1, 2 1

Dept of Biomedical Science University at Albany, State University of New York, 2 Wadsworth Center, New York State Department of Health. Corresponding Author: Dr. Alain Laederach P: (518) 486 4103 F: (518) 474 3181 [email protected]

Chronic obstructive pulmonary disease (COPD) is the fourth highest cause of mortality in the USA. While cigarette smoking is a major cause of COPD, only 15% of smokers develop the disease, indicating major genetic influences. The most widely recognized candidate gene for association with COPD is SERPINA1. Of particular interest is the COPD associated single nucleotide polymorphism (SNP) C116U in the 5’ UTR of SERPINA1. We identified this mutation in a genome wide computational analysis of known disease-associated SNPs that map to human UTRs. What is particularly interesting about this system is that the SERPINA1 5’ UTR has nine alternative splice variants; yet C116U is at least 50 nt away from all splice sites. We therefore postulate that the RNA structure of the 5’ UTR contributes to the COPD association. We computed the partition function of both wild type and mutant RNA for all the splice variants and evaluated their correlation with Pearson coefficient. Using our CAFA approach we experimentally assessed and validated our computational predictions with SHAPE chemical mapping. We ran native gels to observe the folding patterns of the wild type and the mutant; we also performed degradation assays to compare the differences in the rates of degradation of the wild type and the mutants. We observe significant, and splice variant specific effects of the C116U mutation on the 5’ UTR of SERPINA1 confirming the importance of RNA structure in COPD association.

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Role of the Splicing Factor 1 ‘Mystery Domain’ during the Early Stages of Pre-mRNA Splicing Ankit Gupta, Anant A. Agrawal, Karen R. Thickman, Scott D. Kennedy, and Clara L. Kielkopf Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY-14642 Corresponding author: Clara L. Kielkopf – Phone: 585-273-7499, Fax: 585-275-6007 E-mail: [email protected] Splicing Factor 1 (SF1) and U2 snRNP auxiliary factor (U2AF65) form an essential protein complex that recognizes the 3' splice-site during the initial stages of pre-mRNA splicing. A small N-terminal region (ULM) in SF1 is considered to be necessary and sufficient for U2AF65 interactions. A ~100 amino acid domain of SF1 (the ‘mystery’ domain) following the ULM has high sequence conservation from yeast to mammals, yet the function is unknown. Here, we demonstrate that the SF1 mystery domain participates in the SF1/U2AF65 interface and cooperative RNA recognition by SF1/U2AF65 complex. Previously, we observed that heat capacity changes for association of SF1 with the U2AF65-interacting domain (U2AF65-UHM) are greater than those observed with the SF1-ULM. Since heat capacity changes often correlate with the surface area buried by complex formation, that the SF1 ‘mystery domain’ participates in the U2AF65-UHM interface. Now, chemical shift differences among SF1 variants complexed with N15labeled U2AF65-UHM support the hypothesis that the SF1 mystery domain participates in the U2AF65-UHM interface. Isothermal titration calorimetry (ITC) experiments further establish that the SF1 mystery domain contributes to the affinity of SF1 for U2AF65. Significantly, protein/RNA affinity measurements by ITC reveal that the cooperativity of RNA binding by SF1/U2AF65 complex is reduced from a 21-fold enhancement to a 3fold increment by deletion of the mystery domain. We propose that the SF1 mystery domain is important for the proper orientation of the RNA binding domains in SF1/U2AF65 complex relative to the splice site sequences.

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The relaxase of conjugative plasmid pRS01 stimulates retrotransposition of a group II intron residing within the relaxase pre-mRNA Ingrid Hahn, Arthur Beauregard, and Marlene Belfort To date, ~35% of bacterial group II introns have been shown to reside on other mobile genetic elements, which may aid in dissemination. Group II introns retrotranspose to ectopic sites at low frequency. The Ll.LtrB group II intron was originally discovered in the pRS01 conjugative plasmid in the Lactococcus lactis ltrB gene, which encodes a conjugative relaxase. LtrB initiates conjugation by nicking the DNA at the origin of transfer (oriT) sequence. Splicing of the intron in necessary to produce an intact, functional LtrB relaxase, thus allowing conjugative strand transfer. We show here that Ll.LtrB retrotransposition was elevated seven-fold in the presence of an autonomously replicating pRS01 and four-fold in the presence of the LtrB relaxase alone. Second, we demonstrate that catalytic mutants of relaxase hinder retrotransposition. Third, retrotransposition was severely reduced in the presence of bisphosphonates which specifically inhibit relaxase. There results indicate that the LtrB relaxase gene enhances retrotransposition, possibly due to its ability to nick DNA. This implicates a function encoded by a conjugative plasmid as a facilitator of group II intron mobility, providing a rationale for the residence of group II introns on other mobile elements.

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Disease-Associated Mutations that Lead to Structural Rearrangements in Untranslated Regions of RNA Matt Halvorsen, Lauren Neulander, Justin Ritz, Joshua Martin & Alain Laederach† Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, NY 12208 †

To whom correspondence should be addressed. Phone: (518) 486 4103, fax: (518) 474 3181, e-mail [email protected]

Genome-wide association studies (GWAS) often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations we have performed a genome wide analysis of all known diseaseassociated Single Nucleotide Polymorphisms (SNPs) from the Human Gene Mutation Database (HGMD) that map to the untranslated regions (UTRs) of a gene. Rather than using minimum free energy approaches (e.g. mFold), we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. For six disease-states (Hyperferritinaemia Cataract Syndrome, β-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD), and Hypertension) we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the diseaseassociated mutations in the structural ensemble. We observe in several cases (specifically the 5’ UTRs of FTL and RB1) SNP induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a “RiboSNitch,” that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype.

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YrdC exhibits properties of the tRNA modification enzyme, N6 adenosine threonyltransferase Kimberly A. Harris1,3*, Yann Bilbille1, Victoria Jones1, Manal Swairjo2, and Paul F. Agris3 1

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622; 2Western University of Health Sciences, 309 E. Second St. Pomona, CA 91766; 3The RNA Institute, University at Albany, 1400 Washington Ave, Albany, NY 12222 *Phone: 518-437-4448, Fax: 518-437-4456, Email: [email protected] Nucleoside modifications are vital for the proper structure and function of tRNA. The N6-threonylcarbamoyladenosine modification at position 37 (t6A37), 3’adjacent to the anticodon, of many tRNA species in all organisms ensures the accurate recognition of ANN codons by increasing codon affinity and enhancing ribosome binding. Considerable data exists on the biophysical aspects of t6A37. However, the biosynthesis pathway of this hypermodified base is only partially understood. Current information shows this pathway requires ATP, free threonine, a single carbon source, and an unidentified enzyme. Recently, the universal protein family YrdC/Sua5 has been shown to be involved in t6A37 biosynthesis. To further investigate the role of YrdC in t6A37 biosynthesis, we examined the interaction of the E. coli YrdC with the heptadecamer anticodon stem and loop of tRNA lysine (ASLLys). YrdC bound the unmodified ASLLys with high affinity (estimated Kd ≈ 0.27 ± 0.20 µM), as determined by changes of the intrinsic fluorescence of YrdC's two tryptophans. In contrast, the protein bound t6A37-modified ASLLys with significantly lower affinity (estimated Kd ≈ 1.36 ± 0.39 µM). YrdC also demonstrated specificity toward threonine and ATP. The protein’s mechanism of action, studied using NMR, circular dichroism, and fluorescence of 2-aminopuine at position 37, indicated base flipping was not involved. Current data supports the hypothesis that YrdC, alone or as a complex, constitutes the specificities and affinities required of the enzymatic component of t6A37 biosynthesis.

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Targeted 2'-O-methylation at a nucleotide within the pseudoknot of telomerase RNA reduces telomerase activity in vivo Chao Huang and Yi-Tao Yu Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642 Corresponding author: Yi-Tao Yu, Tel: (585)275-1271, Fax: (585)275-6007 Email: [email protected] Telomerase RNA is an essential component of telomerase, a ribonucleoprotein enzyme that maintains chromosome ends in most eukaryotes. Here we employ a novel approach, namely, RNA-guided RNA modification, to assess whether introducing 2'-O-methylation into telomerase RNA can influence telomerase activity in vivo. We generate specific 2'-O-methylation sites in and adjacent to the triple-helix (within the conserved pseudoknot structure) of yeast telomerase RNA (TLC1). We show that 2'-O-methylation at U809 reduces telomerase activity, resulting in telomere shortening, whereas 2'-O-methylation at A804 or A805 leads to moderate telomere lengthening. Importantly, we also show that targeted 2'-O-methylation does not affect TLC1 levels, and that 2'-O-methylated TLC1 appears to be efficiently assembled into telomerase ribonucleoprotein. Our results demonstrate that RNA-guided RNA modification is a highly useful approach for modulating telomerase activity.

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Developing AMPA Receptor Aptamers Zhen Huang, Jae Seon Park, Yan Han, Joe Wang, William Jaremko, Hyojung Seo, Li Niu Department of Chemistry, and Center for Neuroscience Research, University at Albany, SUNY, Albany, New York 12222 In finding new treatment for amyotrophic lateral sclerosis (ALS), one of the important therapeutic strategies is to develop inhibitors of the α-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) receptors. This is because excessive activity of AMPA receptors, generally termed as excitotoxicity, is thought to link to the selective death of motor neurons. We are interested in developing AMPA receptor inhibitors that are both potent and water soluble, the properties superior to all existing inhibitors. Using systematic evolution of ligands by exponential enrichment (SELEX), we have successfully identified three classes of aptamers with nanomolar affinity against AMPA receptors. In the class of competitive aptamers, we found one aptamer with an IC50 value of 30 nM, rivaling any other exiting AMPA receptor inhibitors. Furthermore, this aptamer is broadly active in all AMPA receptor subunits (i.e., GluR1-4), but has no unwanted activity in kainate or NMDA receptors, the two other glutamate receptor subtypes. We have also identified two other classes of noncompetitive aptamers that are differentially selective to conformations of GluR2, a key AMPA receptor subunit that mediates excitotoxicity: one class uniquely inhibits the open-channel whereas the other inhibits the closed-channel conformation. To turn these aptamers into potentially useful drugs, we have now successfully generated a class of chemically modified aptamers that are biostable or resistant with ribonucleases so that these aptamers can be tested in vivo. Our results suggest the possibility of developing aptamers that are nanomolar affinity, water-soluble and highly selective to both an AMPA receptor subunit and a unique receptor conformation. These aptamers are excellent water-soluble, nanomolar affinity templates for design of better inhibitors as drug candidates for potential therapies for ALS and epilepsy.

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Structural Analysis of SLIP1-Mediated Protein-Protein Interaction Networks Patrick Itotia and Roopa Thapar Hauptman-Woodward Medical Research Institute and Department of Structural And Computational Biology, SUNY, Buffalo, NY 14203 Tel: 716-898-8687, Fax: 716-898-8600, E:mail: [email protected] SLBP Interacting Protein 1 (SLIP1) is an essential protein in mammalian cells that has been implicated in histone protein synthesis. SLIP1 binds Stem-Loop Binding Protein (SLBP), a key regulator of histone mRNA metabolism. Intriguingly, RNAi knockdown of SLIP1 results in cell death. This is in contrast to siRNA knockdown of SLBP which only affects cell growth and the cell cycle. Therefore other roles for SLIP1 may exist, besides its involvement in histone protein synthesis. To gain insight into the regulatory pathways and proteinprotein interaction networks that involve SLIP1, we performed a yeast two-hybrid screen to identify SLIP1 interacting proteins. Positive interactions for a subset of identified targets were validated by performing co-immunoprecipitation experiments. Factors involved in histone pre-mRNA processing such as SLBP, 3 hExo; the export factor DBP5, factors that regulate mRNA translation and stability such as the eIF3 complex and the Lsm chaperone complex and the capping factor and oncoprotein c-Myc are also part of the functional network of SLIP1 interacting proteins, suggesting that the protein participates in numerous aspects of RNA-mediated gene expression. To gain structural insight into these interactions, we have co-purifed these interactors with SLIP1 and initiated structural studies on the various complexes using SAXS and X-ray crystallography. Recent progress towards mapping the binding interfaces on SLIP1 using structural and biochemical techniques will be presented.

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Development of the sxRNA platform; A trans-acting RNA switch Sabarinath Jayaseelan, Arthur Beauregard, Francis Doyle and Scott Tenenbaum College of Nanoscale Science and Engineering. Nanobioscience Constellation, University at Albany-SUNY, Albany, New York 12203, USA Naturally-occurring RNA molecules can interact to induce conformational changes, which in turn initiate biochemical reactions and other cellular processes. A significant advantage could be gained if these interactions are harnessed to induce specific cellular processes under particular conditions. We currently are developing a technology termed structurally-interacting RNA (sxRNA). The approach uses an engineered “bait-RNA (sxRNA),” a “triggerRNA” that exists in a cell, and an RNA-binding protein (RBP) that binds to the bait when activated by the trigger. The engineered bait is designed to not form the recognition motif for RBP binding, but will undergo a conformational change in the presence of the trigger-RNA. This conformational change can take many forms, but typically acts as a trigger to activate, suppress, or modulate a cascade of biochemical events, leading to some prophylactic or therapeutic effect or providing a means of diagnosing the presence of a given target polynucleotide. We have identified several naturally occurring bait and trigger-RNAs and have used this information to design custom sxRNAs using a bioinformatic approach. We have currently done in vitro testing on several bait-trigger combinations designed for stem loop binding protein (SLBP) and MS2 bacteriophage protein. In Vitro testing has been optimized based on ribonucleoprotein immunoprecipitation (RIP). Engineered baits show up to fivefold increase in binding to its specific protein in the presence of the designated trigger. Studies of the sxRNA platform will be moved to an in vivo system following in vitro optimization. This platform has potential towards both diagnostics as well as therapeutics.

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High-throughput RNAi screens identify new drug targets for breast cancer Antonis Kourtidis, Cheryl Eifert, Ritu Jain, Jan Baumann, Yan Sun, Margo Gebbie, Jason Wong, Michael Gerdes and Douglas S. Conklin Cancer Research Center, Department of Biomedical Sciences, University at Albany, State University of New York Douglas S. Conklin: PHONE (518) 591-7154; FAX (518) 591-7151; Email: [email protected] Overexpression of the adverse prognostic marker ERBB2 occurs in 30% of breast cancers, however, therapies targeting this gene have not proven to be as effective as was initially hoped. Transcriptional profiling meta-analyses have shown that there are approximately 150 genes co-overexpressed with ERBB2, suggesting that these genes may represent alternative factors influencing ERBB2-positive tumors. We describe an RNA interference-based analysis of these genes that identifies transcriptional regulators of fat synthesis and storage as being critical for the survival of these cells. These transcription factors, Reverbalpha (NR1D1) and PBP, both reside on ERBB2-containing 17q12-21 amplicons and are part of the ERBB2 expression signature. We show that Reverbalpha and PBP indirectly upregulate several genes in the de novo fatty acid synthesis network that is highly active in ERBB2-positive breast cancer cells. MDH1 and ME1, enzymes that link glycolysis and fatty acid synthesis, are also regulated by Rev-erbalpha. The resulting high-level fat production contributes to a cellular physiology based on aerobic glycolysis. Together, these results demonstrate that the cells of this aggressive form of breast cancer are genetically preprogrammed to depend on Rev-erbalpha and PBP for the energy production necessary for survival. Since Rev-erbalpha is also a circadian rhythm regulator, it represents a potential molecular link between the risk factors of diet and “the night shift” in breast cancer. Most importantly, since inhibition of Rev-erbα and PBP specifically increases the sensitivity of ERBB2 overexpressing cells to apoptosis, they are excellent therapeutic targets for this tumor type.

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P3 Domains of Eukaryotic RNases P/MRP: Making a Protein-Rich RNA-Based Enzyme Andrey S. Krasilnikov Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA 16802 (814)865-5050; e-mail: [email protected] Ribonuclease (RNase) P is an essential RNA-based enzyme in all three domains of life; in the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme RNase MRP. Bacterial RNase P holoenzyme consists of a large catalytic RNA component and a small protein. Eukaryotic RNases P/MRP have a substantially more complex organization and consist of a catalytic RNA component and multiple essential proteins. Structural and functional roles of the large protein moiety are not clear. Eukaryotic RNase P RNA and the related RNase MRP have acquired a new structural feature, which is not found in bacteria or archaea: a large helix-loop-helix RNA domain P3. The P3 RNA domain is essential and phylogenetically conserved in eukaryotic RNases P/MRP. It appears to play the role of a hub for the assembly of the large multicomponent protein moiety. To shed light on the structural organization of eukaryotic RNases P/MRP, we have crystallized a complex of the yeast P3 RNA domain with RNase P/MRP protein components Pop6 and Pop7, and solved its structure to 2.7A. The structure reveals the fold of the well-structured P3 domain RNA, which is involved in extensive interactions with the proteins. The two proteins form a heterodimer and adopt similar Alba folds. This is the first structure of any part of eukaryotic RNases P/MRP. It provides the first view of RNA-protein interactions in RNase P and suggests roles for this key domain of complex eukaryotic ribozymes.

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Pin1 Regulates mRNA Turnover of Histone mRNAs And AREcontaining mRNAs Nithya Krishnan and Roopa Thapar Hauptman-Woodward Medical Research Institute and Department of Structural And Computational Biology, SUNY, Buffalo, NY 14203 Tel: 716-898-8687, Fax: 716-898-8600, E:mail: [email protected] Many proteins involved in posttranscriptional control of gene expression are regulated by reversible posttranslational modifications such as phosphorylation and ubiquitination. The mechanisms by which these modifications influence gene expression are not well understood. Replicationdependent histone mRNAs are the only mRNAs that have no introns or poly(A) tail and are tightly regulated during the S-phase of the cell cycle. They have a conserved stem-loop in the 3’ UTR that binds Stem-Loop Binding Protein (SLBP). The SLBP RNA-binding domain undergoes phosphorylation-directed prolyl isomerization and forms a specific complex with the prolyl isomerase Pin1 in vitro and in vivo. We will report that Pin1 can act in concert with the protein phosphatase PP2A to dissociate the highly stable SLBP-histone mRNA complex. RNAi knockdown of Pin1 stabilizes SLBP in vivo and inhibition of PP2A in these cells results in accumulation of ubiquitinated SLBP in the nuclear periphery. Our microarray and qPCR analysis of Pin1 RNAi treated cells shows that Pin1 regulates the stability of a subset of mRNAs that include histone mRNAs and ARE-containing mRNAs. Pin1 has previously been shown to modulate the activity of the RNA Polymerase II CTD by stimulating CTD hyperphosphorylation. In yeast, Ess1 (Pin1) has been shown to be important for transcription initiation and termination via a functional interaction with Ssu72, Pta1, and TFIIB likely via gene looping. Our results suggest that Pin1 may play a general role in modulating the stability and phosphorylation state of RNA processing factors thereby influencing the transcription cycle, mRNA export, or turnover.

P-32

Mis-localization of Arp2 mRNA impairs persistence of directional cell migration Guoning Liao, Brittany Simone+ and Gang Liu* Center for Cell Biology & Cancer Research, + summer research program, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208 * Corresponding author: phone: 518-262-9098; Fax: 518-262-5669; E-mail: [email protected] Arp2/3 complex is an actin polymerization nucleator and localized in the leading protrusions of migrating cells. It has been unclear how this complex is targeted to the protrusions and whether its localization is functionally important. We previously demonstrated that mRNAs encoding for the seven subunits of the complex were localized in the protrusions of fibroblasts, suggesting a mechanism to target the complex to the protrusions. We here present data demonstrating the importance of Arp2/3 complex mRNA localization in directional cell migration. Using a recently identified novel mechanism by which Dia1 mRNA is targeted to the perinuclear region, we re-directed the mRNA encoding Arp2, a subunit of the Arp2/3 complex, to the perinuclear region in fibroblasts. We first confirmed the role of Arp2/3 complex in normal protrusion formation and cell migration by showing that knockdown of Arp2 resulted in narrow protrusions and altered cell migration behaviors including loss of directionality. Under the conditions of Arp2 knock-down, re-expression of a protrusion-localizing Arp2 mRNA restored the loss of directionality. In contrast, re-expression of a mis-localizing Arp2 mRNA could not restore directionality. These results demonstrate that localization of Arp2/3 complex mRNAs in the leading protrusions is functionally important for directional cell migration.

P-33

A RNA zipcode independent mechanism that localizes Dia1 mRNA to perinuclear ER via interactions of Dia1 nascent peptide and Rho-GTP Guoning Liao, Xinghong Ma and Gang Liu* Center for Cell Biology & Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208 * Corresponding author: phone: 518-262-9098; Fax: 518-262-5669; E-mail: [email protected] Signal peptide-mediated ER localization of mRNAs encoding for membrane and secreted proteins and RNA zipcode-mediated intracellular targeting of mRNAs encoding for cytosolic proteins are two well-known mechanisms for mRNA localization. Here we report a previously unidentified novel mechanism by which mRNA encoding for Dia1, a cytosolic protein without a signal peptide, is localized to the perinuclear ER in a RNA-zipcode-independent manner in fibroblasts. Dia1 mRNA localization is also independent of actin and microtubule cytoskeleton but requires translation as such that the association of Dia1 nascent peptide with the ribosome-mRNA complex is necessary. Sequence mapping results indicate that interactions of the GTPase binding domain of Dia1 peptide with active GTPase Rho is important for Dia1 mRNA localization, providing the first evidence that GTPase Rho plays a direct role in mRNA localization. This mechanism can override beta-actin mRNA zipcode and re-direct beta-actin mRNA from protrusions to the perinuclear region, providing a new way to manipulate intracellular mRNA localization.

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What drives condensation of double-stranded RNA Li Li, Suzette A. Pabit, Steve P. Meisburger, and Lois Pollack School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA Phone: 6072558695, Fax: 6072557658, email: [email protected] DNA condensation is of great interest due to its fundamental biological importance. With the discovery of the important roles of RNAi, recent attention has been focused on efficient packaging of dsRNA for therapeutics. In this study, we applied UV spectroscopic and small angle x-ray scattering to investigate the mechanism of RNA condensation. Our results suggest that, under conditions where DNA duplexes significantly precipitate, identically charged RNA duplexes do not though they associate via end-to-end stacking. The forces that lead to side-by-side attraction and subsequent condensation may be highly correlated with the differing geometric property of RNA and DNA.

P-35

POSTER SESSION II 10:00 am – 11:00 am Saturday, Oct 9, 2010

Effect of Mg2+ ions on mechanical unfolding of a two-base-pair RNA kissing hairpin Pan T. X. Li Department of Biological Sciences, SUNY Albany Structure and function of RNA critically depend on ionic conditions. However, rigorous thermodynamic analysis of metal ions binding to polyelectric RNA remains a daunting task. To reduce complexity of this problem, we examined folding of a kissing complex with only two base pairs formed between two hairpins. Using optical tweezers, single kissing RNA molecules are stretched and relaxed from 5'- and 3'-ends. We can monitor folding of the kissing interaction in real time and distinguish its force and extension signals from those of secondary structure. We studied folding of this kissing complex under various Mg2+ ion concentrations. Mg2+ ions stabilize the kissing complex and speed its folding. However, Mg2+ ions have distinct effects on forward and reverse reactions. At saturation concentration of Mg2+ ions, the two-base-pair kissing complex breaks at >40 pN, whereas kissing forces merely change by ~5 pN. We will discuss how equilibrium, kinetic barrier and work dissipation change with concentration of Mg2+ ions and applied force. We will also discuss retrieval of equilibrium free energy under non-equilibrium pulling conditions.

P-36

UAlbany Proteomics/Mass Spectrometry Core Qishan Lin and Jinghua Zhu Cancer Research Center, University at Albany, One Discovery Drive, Rensselaer, NY 12144 Phone: 518-591-7214; Fax: 518-591-7211; E-mail: [email protected] The Proteomics Facility was launched in the year of 2002. The Facility provides researchers with access to the state-of-the-art mass spectrometric instrumentation for bio-molecular (RNA, DNA and protein) identification and quantification. The Core offers high-throughput proteomics technologies and platforms for the discovery, quantification, and verification of biomarkers for various diseases; and regularly assists researchers in studying animal models of human diseases, or with the characterization of disease states like infectious disease, cancers, neurodegenerative diseases and human ageing. During the past few years, the Facility has established successful collaborations with, and performed work on a fee-for-service basis for more than 300 clients around the world that include major research institutions, government agencies, scientific organizations, and pharmaceutical companies. The Facility delivers definitive, high quality results in a time and cost effective manner with significant emphasis on client service and value-added solutions. Typical services include but not limit to 1) liquid chromatography, 2) RNA, DNA and protein purification, 3) gel electrophoresis and imaging analysis, 4) sample processing (isotope labeling, enzymatic digestion and fractionation), 5) MALDI-TOF peptide mass fingerprint (PMF), 6) phosphopeptide mapping (TiO2, IMAC, LC-MS/MS approach), 7) protein expression profiling through MudPIT, iTRAQ, or SILAC, 8) protein biomarker discovery, 9) metabolite id and quantitation, and 10) de novo peptide sequencing. Specific services provided by the Core are outlined in our poster. For more information on services, prices and sample submission procedures, contact Qishan Lin at [email protected]

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Thermodynamic characterization of an unusual pseudoknot from influenza A virus RNAs Fei Liu, Carla A. Theimer Department of Chemistry, State University of New York at Albany The influenza A virus is responsible for seasonal flu epidemics and reoccurring pandemics, resulting in a worldwide threat to public health. H5N1, a highly pathogenic avian influenza A virus, caused disease outbreaks among poultry in southeast Asia (late 2003 to early 2004) and the same virus was fatal to humans in Thailand and Vietnam. More recently in 2009, concerns have increased due to the emergence of a pandemic caused by a new strain of the influenza A (H1N1) virus. The reason why these more recent H5N1 and H1N1 strains are more aggressive than previously circulating strains is still unclear. Since RNA structure plays an important role in the life cycle of RNA viruses, the higher-order structure of the influenza virus RNA deserves further investigation. Recent studies have identified a conserved structure in the coding region of segment 8 from type A influenza viruses and demonstrated that a unique single nucleotide substitution within the conserved structure, typically associated with recent H5N1 strains, dramatically affects the equilibrium between a hairpin and an unusual pseudoknot conformation near the 3’-splice-site of the NS gene. This conformational shift was proposed to have consequences for the splicing regulation of the segment 8 mRNA. In this study, UV melting and native gel electrophoresis have been used to examine the thermodynamic stability differences between RNA constructs designed to probe the proposed conformational equilibrium. Ongoing studies are investigating the connections between the RNA structures, the conformational equilibrium, and splicing efficiency.

P-38

Using Aptamer-derived Molecular Adaptors to Commandeer a Biological Pathway Prabhat K. Mallik, Kimi Nishikawa, Albert J. T. Millis, and Hua Shi Department of Biological Sciences, University at Albany, State University of New York, Albany, New York 12222, USA Biological processes are governed by specific molecular interactions, and induction of molecular proximity can mediate a discrete functional response. Therefore, creating new molecular connectivity between non-interacting proteins is a means of imposing rational control over biological processes to serve experimental or therapeutic purposes. Here we use composite RNA aptamers to create molecular adaptors that can link various “target” molecules to a common “utility” molecule. In this configuration, the utility molecule is an entry point to a pathway being conscripted to process the target molecule. To prove this principle, we created a bi-functional aptamer that simultaneously binds to the green fluorescent protein (serving as a surrogate extracellular target protein) and the opsonin C3b/iC3b (serving as the utility molecule). This bifunctional aptamer enabled us to commandeer the C3-based opsonization-phagocytosis pathway to specifically transport an extracellular target into the lysosome for degradation. This novel strategy has the potential for powerful therapeutic applications with extracellular proteins involved in tumor development or surface markers on cancer cells as the target molecules. Opsonization is an important immunological mechanism of host defense, in which foreign substances are tagged by opsonin molecules for destruction. The most important opsonins are antibodies (especially IgG) and complement proteins such as C3b and its closely related fragment iC3b. These opsonins act as adaptors to connect a wide variety of target particles with a few common receptors on effector cells such as macrophages or natural killer (NK) cells. Phagocytosis by macrophages and other professional phagocytes is one of the major consequences of opsonization, in which particle internalization is initiated by the receptor-opsonin interaction. Because opsonization is a powerful and efficient way of eliminating harmful and/or unwanted particles, it would be desirable to re-direct this mechanism at will to selectively destroy disease-causing molecules or cells, especially those that are not recognized by the immune system as “foreign.” In this study, we attempt to commandeer C3b/iC3b molecules to destroy or damage the intended targets. We equiped C3b/iC3b with an adaptor providing high specificity and efficiency, thereby enabling us to intentionally tag unwanted “self” proteins or cells as “foreign” to elicit a response against them. We envision this molecular adaptor for C3b/iC3b as a composite bi-functional aptamer. Each adaptor comprises at least two individual aptamers, one for a predetermined target molecule and one for C3b/iC3b, so that both the target and the opsonin can simultaneously bind to the adaptor to form a triple complex. The C3b/iC3b molecule in the triple complex will be recognized by its receptors on the surface of the effector cells. The strategy presented here would augment the potency of target-binding aptamers so that the targets are not neutralized reversibly, but destroyed or damaged irreversibly. Two major challenges are involved in implementing this strategy: (1) finding the ideal “utility” aptamer that binds to C3b/iC3b with proper affinity without inhibiting its opsonizing function, and (2) building the composite aptamer in which both the targeting aptamer and the utility aptamer are folded properly. We have successfully identified aptamers for the complement component C3 and its activated derivatives, C3b/iC3b, and constructed a bi-functional aptamer using one of these as the “utility moiety.” To establish a straightforward functional assay to prove the principle, we chose the green fluorescent protein (GFP) as a surrogate target, because this protein can be visualized in its route through the opsonization-phagocytosis pathway.

P-39

Visualizing RNA Secondary Structure Changes Using PCA Joshua S. Martin, Matt Halvorsen & Alain Laederach† Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, NY 12208 †

To whom correspondence should be addressed. Phone: (518) 486 4103, fax: (518) 474 3181, e-mail [email protected]

We have identified over 50 disease-associated single nucleotide polymorphisms (SNPs) in the intergenic and non-coding regions of the human genome that significantly change the ensemble of RNA conformations from wild-type. We describe here a unique way to characterize and visualize the nature of the conformational changes induced by the disease-associated mutants using the 5' UTR of FTL as an example. Our method utilizes principal component analysis (PCA) to visualize an ensemble of structures sampled from a Boltzmann distribution. This ensemble of structures undergoes a conformational change with disease-associated SNPs which are analogous to that observed in bacterial regulatory Riboswitches when specific ligands bind. The mathematical nature of our representation allows for the easy projection of chemical mapping data onto the graphs to visualize the most likely structures found in solution.

P-40

Testing the affect of retrotransposition on genome instability during yeast aging Patrick H. Maxwell1*, William C. Burhans2, & M. Joan Curcio1 1

Laboratory of Molecular Genetics, Wadsworth Center and Department of Biomedical Sciences, University at Albany School of Public Health, Albany, NY 2 Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY *phone: 518-486-3821, fax: 518-474-3181, e-mail address: [email protected] While mutations and chromosome rearrangements increase with age in humans and model organisms, genome instability has not yet been demonstrated to play a causative role in normal aging, and specific mechanisms responsible for agingassociated genome instability have not been fully elucidated. Retrotransposons are mobile genetic elements found in many eukaryotic genomes that replicate through an RNA intermediate. Integration of new copies of retrotransposons into genomes (retrotransposition) can cause mutations in genes, and we and other groups have identified associations between retrotransposons and chromosome rearrangements. We are testing whether retrotransposition promotes agingrelated genome instability by studying Ty1 retrotransposons in Saccharomyces cerevisiae using a chronological aging model, which examines the survival and characteristics of yeast cells maintained in stationary phase cultures. We examined cell viability and loss of heterozygosity frequencies in diploid strains with or without mutations that down-regulate Ty1 retrotransposition and wild-type diploid strains grown in various media that increase or decrease Ty1 retrotransposition. Higher levels of Ty1 retrotransposition were correlated with decreased viability and increased loss of heterozygosity frequencies. We did not observe an increase in Ty1 integration at a known integration hot-spot with increased chronological age, indicating that any direct role for Ty1 in aging would likely result from retrotransposition that occurs prior to stationary phase. Future work will further explore whether Ty1 is directly contributing to aging and genome instability in yeast.

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Turning up the heat: large favorable enthalpy changes drives RNA recognition by RNA recognition motif (RRM)-containing proteins Krystle J. McLaughlin, Zhenjiang Xu, Jermaine L. Jenkins, Clara L. Kielkopf Department of Biochemistry and Biophysics, University of Rochester Medical Center Clara L. Kielkopf: 585-273-7499, fax-585-275-6007 [email protected] Little is known concerning the thermodynamic basis for RNA recognition by RNA recognition motifs (RRM), a highly prevalent class of eukaryotic RNA binding domain. Here, we reveal that unusually large enthalpy changes drive RNA binding by four distinct RRM-containing proteins. These include three pre-mRNA splicing factors that specify uridine-rich sites: (i) U2AF65, an essential pre-mRNA splicing factor that recognizes constitutive 3' splice site signals; (ii) SXL, a wellcharacterized alternative splicing factor that antagonizes U2AF65; (iii) TIA-1, an alternative splicing factor that promotes use of specific 5' splice sites. For contrast, the fourth RRM-containing protein (iv) polyadenosine binding protein (PAB) binds a purine tract and is involved in mRNA translational control. All four RRM-containing proteins exhibit remarkably large magnitudes for the enthalpy and the entropy changes during RNA binding. Several potential sources of this thermodynamic signature are investigated. Experiments with SXL in a series of different buffers rule out the possibility that multiple protonation events are coupled to RNA binding. Likewise, polyadenosine compared with polyuridine titrations of SXL and PAB rule out the possibility that base-base stacking of polypurines significantly reduces an inherently large enthalpy-entropy change of RNA binding. However, the enthalpic and entropic contributions were reduced substantially during non-specific binding of polyadenosine by SXL or polyuridine by PAB. Further, site-specific mutations of ribonucleoprotein motifs implicate conserved aromatic residues of the RRM as key contributors to this thermodynamic effect. Altogether, these results suggest unusually large enthalpy and entropy changes are a general characteristic of site-specific RNA recognition by RRM-containing proteins.

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Ribonuclease III of the bacterial extremophile, Thermotoga maritima. Catalytic behavior, and analysis of the reactivity epitopes of the cognate pre-rRNA substrates. Lilian Nathania* and Allen W. Nicholson Temple University Department of Chemistry 1901 N. 13th Street, Philadelphia, PA 19122 Phone: (215) 204-4903 Fax: (215) 204-1532 [email protected] Structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. However, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. The biochemical properties of purified Thermotoga maritima (Tm) RNase III were analyzed using the cognate pre-ribosomal RNAs as substrates. Tm-RNase III catalytic activity is dependent upon divalent metal ion, with Mg2+ as the preferred species, and is also supported by salt (Na+, K+ or NH4+), with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70˚C, with significant activity retained up to 95˚C. The T. maritima genome encodes a ~5,000 nt transcript, expressed from the single ribosomal RNA (rRNA) operon. The RNase III processing sites are formed through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The pre-16S and pre-23S processing stems were synthesized as small hairpin RNAs, and were site-specifically cleaved by Tm-RNase III to produce the immediate precursors to the mature rRNAs. The processing reactivities of specific base-pair (bp) sequence variants of the Tm pre-23S RNA hairpin indicates a strong dependence of processing reactivity on the bp sequence within the 4 bp proximal box (pb). The preferred bp sequence in the pb is similar to that of in Escherichia coli (Ec) RNase III substrates, and Tm-RNase III cleaves an EcRNase III substrate with identical specificity. These studies reveal a broad phylogenetic conservation of substrate reactivity epitopes among bacterial RNases III.

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Confirming RNA Structure Change Predictions Using SHAPE Reactivity Data Lauren Neulander, Justin Ritz, Matt Halvorsen & Alain Laederach† Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, NY 12208 †

To whom correspondence should be addressed. Phone: (518) 486 4103, fax: (518) 474 3181, e-mail [email protected]

We have identified several disease-associated single nucleotide polymorphisms (SNPs) in the human genome that alter the structure of a regulatory UTR. We focus here on the 5’ UTR of the FTL (Ferritin Light Chain) gene. Mutants in the 5’ UTR of the FTL gene have been shown to cause Hyperferritinemia Cataract Syndrome, a disease that results in the crystallization of ferritin in the eye. We describe a unique way to identify and visualize the conformational changes induced by the disease-associated mutations. Using the SNPfold algorithm, we have identified several mutations of the FTL gene that significantly alter the structure of the wild-type UTR. In most of these cases destabilization of the iron response element is predicted. Based on these computational analyses, we then did a comparison of the chemical mapping data of five mutants, two controls and the wild-type RNA to confirm changes in accessibility at each nucleotide in the sequence.

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Contributions of Helix Topology and the Counterion Cloud in RNA Interactions Suzette A. Pabit*, Li Li, Steve P. Meisburger and Lois Pollack School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853 *Corresponding author: 607-255-6408 (telephone), 607-255-7658 (fax) and [email protected] (e-mail address) It is universally accepted that RNA molecules are more foldable and adapt more conformational structures than the rigid uninterrupted double helix of DNA molecules. The role of metal ions in RNA folding interactions is well documented. Divalent ions like Mg2+ are very effective in stabilizing RNA tertiary structures; and specific binding sites for Mg2+ ions has been found in crystallographic studies of folded RNA. Unfortunately, previous studies focused on specifically bound or chelated ions in pockets of RNA structure and the role and distribution of diffuse counterions in stabilizing and promoting the diversity of RNA structures has not been as thoroughly investigated. Using empirical approaches from Smallangle X-ray Scattering (SAXS) measurements, we investigated the role of monovalent and divalent ions in counterion-mediated charge screening efficiency of short double-stranded DNA and RNA helices. Numerical calculations based on the Nonlinear Poisson Boltzmann (NLPB) equation then allowed us to visualize ion distributions around the helices. We find that in both monovalent and divalent ion environments, charge is more effectively screened in RNA than in DNA with divalent ions providing significantly stronger screening effect than monovalents even in the absence of specific divalent binding sites. Closer ion association and localization in the deep major groove of RNA leads to a more efficient charge screening which could be important in selective recognition of RNA binding partners, RNA structure versatility and RNA folding.

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Ribosome-Associated Factors Regulate the Ty1 Retrotransposon Ryan J Palumbo◊‡, Lauren J Neulander-Davis‡, Justin Ritz‡, Alain Laederach◊‡, and M. Joan Curcio◊‡* ◊The State University of New York at Albany School of Public Health Biomedical Sciences Department ‡The New York State Department of Health Wadsworth Center *Corresponding author. Telephone: 1-518-473-6078. Fax: 1-518-474-3181. Email: [email protected] The yeast Saccharomyces cerevisiae harbors 78 known ribosomal protein genes, 57 of which have a paralog—an identical or nearly identical copy. The paradigm that these paralogs have been conserved solely to confer a dosage benefit has been challenged by recent work which suggests that a subset of the 57 ribosomal protein paralogs (RPPs) have evolved independent, paralogspecific functions. Using the endogenous Ty1 retrotransposon as a model, we show that retromobility is reduced in both a paralog-specific manner—where a deletion of one RPP gene more severely affects mobility than a deletion of its corresponding paralog; and in a paralog-dependent manner—where both Ty1 protein and mobility are abolished in one rpp∆, but elevated when its corresponding paralog is deleted. We also show that Ty1 mobility is dependent on a set of ribosome biogenesis factors (RBFs). The majority of RPPs and RBFs promote Ty1 retrotransposition at a post-transcriptional step and at least two are required for efficient Ty1 protein synthesis. Our data suggest that specific RPPs and RBFs may impact the generation of transcript-specific ribosomes, whereby the combination of paralogs present in any given ribosome determines the strength by which certain transcripts are targeted and translated. Our recent work suggest that the 5’ end of the Ty1 RNA is highly structured, and we will present results of experiments to determine whether the structure is involved in translational regulation of the Ty1 transcript.

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Markers of translational infidelity and ER stress are associated with deficiencies in Trm9-catalyzed tRNA modifications Ashish Patil, Madhu Dyavaiah, John Rooney, Urlike Begley & Thomas Begley Department of Biomedical Sciences, Cancer Research Center, University at Albany, State university of New York, Rensselaer, NY 12144 Corresponding author: 518-591-7163, [email protected] The Saccharomyces cerevisiae methyltransferase 9 (Trm9) methylates the wobble uracil of specific tRNAs to generate (mcm5U34) 5methylcarbonylmethyluridine and 5-methylcarbonylmethyl-2-thiouridine (mcm5s2U34) modifications. We have demonstrated that the methylation of the wobble uracil by Trm9 enhances the translation of some codon specific transcripts (YEF3, RNR1, and RNR3), with this increase in protein levels independent of transcription. Wobble base modifications similar to those catalyzed by Trm9 have previously been implicated in preventing translational errors. In accordance the trm9 cells were sensitive to translational error inducers like Paromomycin. We used reporters, western blots and northern blots to demonstrate increased amino acid misincorporation and increased translational error markers in trm9 mutants. Our UPR (Unfolded Protein Response) reporter assay demonstrated an increased unfolded protein response in trm9 cells, which was further induced on treatment with paromomycin. Our northern blot results showed increased basal and paromomycin induced levels of UPR markers like KAR2 and HAC1Spliced transcripts in trm9 mutants. Ultimately, our study supports that the reduced translational fidelity leads to the increased UPR in the trm9 mutants.

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Viability of a Mouse Sarcoma in the Absence of Dicer Arvind Ravi1,2, Madhu S. Kumar1, Christine Chin1, Tyler Jacks1,3, Phillip A. Sharp1† 1

MIT Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts 02139, USA. 2Harvard-MIT Health Sciences and Technology Program, Cambridge, Massachusetts 02139, USA. 3Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. †

To whom correspondence should be addressed. Email: [email protected], Phone: 617-253-6421, Fax: 617-253-3867

MicroRNAs (miRNAs) are short approximately 23 nucleotide RNAs thought to collectively regulate thousands of targets within the genome. While over half of all mammalian transcripts are predicted to be under pressure to maintain target sites for these short RNAs, individual miRNA-target interactions validated in vitro often show only modest translational repression, suggesting that miRNAs may simply be fine-tuning gene expression. To better evaluate the global role of miRNAs in cellular processes, we studied a mouse sarcoma following loss of Dicer, a critical enzyme required to generate miRNAs. In contrast to many other somatic cell models, Dicer loss had seemingly little consequence, and a majority of isolated clones had undergone complete recombination. Not only were Dicer null sarcoma cells capable of being passaged indefinitely, but perhaps even more surprisingly, they retained the ability to form subcutaneous tumors. Although relatively similar to their wild-type counterparts under basal conditions (albeit with a mild proliferative defect), Dicer null cells were more susceptible to cellular stressors such as exposure to sodium arsenite. Consistent with pathway analysis results suggesting altered redox regulation, in vitro testing identified Bach1, a key inhibitor of the oxidative stress response, as a direct let-7 target. The model presented here contradicts an essential role for miRNA function in certain cellular states and offers a unique opportunity for further study of the removal of essentially an entire regulatory level in mammalian cells.

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Reconstruction of RNA tertiary structures from predicted secondary structures and RNA 3D motifs Vladimir Reinharz, Jérôme Waldispuhl School of Computer Science & McGill Centre for Bioinformatics, McGill University, 3480 University Street, Montreal, Quebec H3A 2A7, Canada * Corresponding author: Phone: +1-514-398-5018, Fax: +1-514-398-3883, Email: [email protected] The prediction of RNA three-dimensional structures from its sequence only is a milestone to RNA function analysis and prediction. In recent years, many methods addressed this challenge, ranging from cycle decomposition and fragment assembly to molecular dynamics simulations. However, these techniques are heavily dependent on the reliability of the scoring schema and their predictions remain fragile. In particular, the energetic contribution of secondary structure interactions (i.e. Watson-Crick and Wobble base-pairs) is now well documented, but the quantification of noncanonical interactions – those shaping the RNA tertiary structure – is poorly understood. Nonetheless, even if a complete RNA tertiary structure energy model is currently unlikely, we now have catalogues of recurrent local 3D structural motifs, including non-canonical base pairings, found in experimental structures. A practical objective is thus to develop techniques enabling to use this knowledge for robust RNA tertiary structure predictors. In this work, we benefit of the progresses accumulated over the last 30 years in the field of RNA secondary structure prediction and expand these methods to incorporate the novel local motifs information available in databases. Using a integer programming framework, our method modifies predicted secondary structures (i.e. add or remove canonical base-pairs) to accommodate the insertion, inside the backbone, of the best fitted RNA 3D motifs. We describe our algorithm, discuss the complexity of insertion of different classes of motifs, harvested in the H. marismortui, and experiment our approach on a test-set of representative ncRNAs.

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RNAstructure: Software for RNA secondary structure prediction and analysis Jessica S. Reuter and David H. Mathews Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, NY 14642, USA Phone: (585) 683-7329 Fax: (585) 275-6007 Email: [email protected] To understand an RNA sequence's mechanism of action, the structure must be known. Furthermore, target RNA structure is an important consideration in the design of small interfering RNAs and antisense DNA oligonucleotides. RNA secondary structure prediction and analysis, using thermodynamics, can be used to develop hypotheses about the structure of an RNA sequence. RNAstructure is a software package for RNA secondary structure prediction and analysis. It uses thermodynamics and utilizes the most recent set of nearest neighbor parameters from the Turner group. It includes methods for secondary structure prediction (using several algorithms), prediction of base pair probabilities, bimolecular structure prediction, prediction of structures common to multiple sequences, secondary structure visualization, and structure comparison. The package includes a library of C++ classes for incorporation into other programs, a user-friendly graphical user interface written in JAVA, and new Unixstyle text interfaces. The original graphical user interface for Microsoft Windows is still maintained. The extensions to RNAstructure serve to make RNA secondary structure prediction and analysis user-friendly. The package is available for download from the Mathews lab homepage at http://rna.urmc.rochester.edu/RNAstructure.html.

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Towards high-throughput structural mapping of biopolymers by pyrite footprinting 1

2

2

1

Jörg Schlatterer , Chris Jones , Lois Pollack and Michael Brenowitz 1

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461

2

School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853

High-throughput structure determination of DNA, RNA and proteins, is a key component of structural genomics where many structures and/or complexes need to be interrogated. Hydroxyl radical (·OH) ‘footprinting’ leverages the small size and high reactivity of ·OH to produce solvent accessibility maps of macromolecules with as fine as single residue resolution. These maps can be used as constraints during structural modeling of biopolymers and their complexes and thus contribute to the illumination of structural folds and general architecture. Commonly used methods of ·OH generation are not amenable to high throughput footprinting. Geochemical studies showed the potential of iron disulfide (pyrite) to generate ·OH in aqueous solutions. We are designing and fabricating devices using pyrite as a solid phase matrix for high-throughput ·OH ‘footprinting’ analysis of DNA, RNA, and proteins (Patent application pending). Pyrite particles with defined particle sizing are ground from mineral chunks. Standardized pyrite powder and double stranded DNA are incubated in aqueous solution in a microfluidic device by continuous flow technology. DNA fragment detection is achieved by primer extension and capillary electrophoresis. Cleavage profiles with single-nucleotide resolution correspond to results derived from standard Fenton reaction generated ·OH. Parallel arrays of microfluidic devices will allow efficient analysis of large numbers of samples.

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Alternative functional significance of alternative splicing Adonis Skandalis and Emma Bondy-Chorney Brock University St. Catharines, Ont. Canada Corresponding author: Adonis Skandalis, Tel. 905-688 5550 telephone e-mail [email protected] In order to elucidate the biological significance of unproductive alternative transcripts and to investigate the mechanisms underlying the fidelity of mRNA splicing we have analysed the comparative evolutionary conservation of unproductive splice variants of select housekeeping genes among diverse mammalian phylogenetic groups. Our analyses of hundreds of alternative transcripts in the indicator loci POLB and MLH1 indicate that here is little evolutionary conservation of splice variant types or overall frequency among mammals, birds or even among primates. The frequency of unproductive alternative splicing, however, is highly and positively correlated to maximal lifespan, but not to metabolic rate. While these observations indicate that individual splice variants may not be functional they support the notion that alternative splicing may have an important biological role. In this presentation we will present a model on the nature of this role.

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Thermodynamic Studies of the PreQ1 Riboswitch Nakesha L. Smith, Carla A. Theimer Department of Chemistry, State University of New York, 1400 Washington Ave., Albany, NY 12222 518-591-8875 (p), 518-442-3462 (f), [email protected] Bacteria use RNA riboswitches to control the expression of key metabolic genes in response to chemical factors in their environment. Riboswitches are naturally occurring regulatory RNA structures typically found in the 5’ untranslated regions of certain bacterial (and some eukaryotic) messenger RNAs. Although the mechanism has not been fully characterized, naturally occurring riboswitches have been shown to control metabolic gene expression in a number of medically relevant bacteria. The preQ1 riboswitch regulates gene expression in response to preQ1, the biosynthetic precursor of queosine, an essential hypermodified guanine nucleotide in the tRNAs of a number of amino acids. This switch represents a good model system for biophysical characterization due to its relatively small size and the availability of high resolution structural information for the ligand-bound aptamer domain. Recent NMR and X-ray crystallographic studies revealed an Htype RNA pseudoknot that sequesters a portion of the 3’ A-rich tail that would otherwise form a transcriptional anti-terminator stem. In this study, the B. subtilis preQ1 riboswitch was investigated using optical melting, NMR spectroscopy, and native gel electrophoresis in order to examine the overall structure-function-stability relationships of entire riboswitch.

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The Yersinia pestis RNA Chaperone Hfq Is an Essential Component for Surviving Changing Environmental Conditions. Kris E. Spaeth, Guangchun Bai, Eric Smith and Kathleen A. McDonough* Wadsworth Center, Albany, NY *Corresponding author: (518) 486-4253, [email protected] The life cycle of Yersinia pestis exposes the bacteria to substantially different environmental temperatures due to its cycling between fleas (26°C) and mammalian host organisms (37°C). The use of small RNAs (sRNA) to posttranscriptionally alter gene expression is one mechanism commonly used by many bacteria to handle extracellular stresses. The RNA chaperone Hfq plays a central role in enabling sRNA regulation of gene expression in many bacteria species. In preliminary studies done in our lab, we used homologous recombination to delete hfq from Y. pestis Kim6+ and observed that this deletion strain was defective for growth at 37°C. To further explore how Hfq contributes to the biology of Y. pestis we exposed the Δhfq strain to a wider range of temperatures and a variety of extracellular stresses such as oxidative, osmotic and cell wall integrity stresses. The loss of Hfq in Y. pestis decreased the viability of the bacteria at temperatures above 30°C and increased sensitivity to oxidative stresses caused by hydrogen peroxide. The most striking phenotype observed in the absence of Hfq was temperature sensitivity so we focused on further characterizing this defect. Fluorescence microscopy was used to examine the mutant’s morphology at the non-permissive growth temperature. Loss of Hfq caused Y. pestis to form hyper-extended rods at 37°C. Furthermore, analysis of the bacterial DNA content showed that these elongated bacteria contained several condensed chromosomes per cell. These data support a role for sRNA in Y. pestis growth. Further studies are needed to define the specific roles of Hfq and sRNAs in Y. pestis biology.

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Improving the Amber RNA force field Aleksandar Spasic,1 Alan Grossfield,1 Harry Stern,2 David Mathews1 1

Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, NY 14642 2Department of Chemistry, University of Rochester,RC Box 270216, Rochester, NY 14607 Corresponding author: Aleksandar Spasic, Phone: (585) 276-4338, FAX: (585) 275-6007, Email: [email protected] We describe a new method for improving the parameters of the Amber force field for RNA by fitting to energies and forces from quantum chemistry calculations. Parameters for the Amber force field currently in use were obtained from sources such as vibrational frequencies and crystal data for bonded and van der Waals parameters and QM calculations for the partial charges. We are pioneering a new approach in which we keep the Amber force field functional form but fit all the parameters simultaneously to reproduce the energy and forces determined by a high level density functional theory (DFT) calculation. The procedure is as follows. First, we assemble a collection of structures which are used for fitting. We use 10,000 two-nucleotide structures which were obtained from crystal structures and molecular dynamics. Half of these structures are covalently bonded and half are hydrogen-bonded. Next, we perform B97D/aug-cc-pVTZ calculations on these structures to calculate their energies and forces. Finally, we perform a two stage fit to determine the parameters. In the first phase, a linear least-squares fit for the bonded force constants and the coefficient of r-12, r-6, and r-1 terms for pairs of atom types is performed. In the second phase, these pair coefficients are then used as target data for optimizing charges and Lennard-Jones parameters using a non-linear least-squares fit. Comparison of Amber energies with the fitted parameters shows that the new parameter set is capable of reproducing the DFT energy landscape better that the original parameter set.

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A hierarchical model for evolution of ribosomal RNA Sergey V. Steinberg * and Konstantin Bokov Département de Biochimie, Université de Montréal PO Box 6128, Station Centre-ville, Montréal (QC) H3C 3J7, Canada Tel: 514-343-6320, fax: 514-343-2210, E-mail: [email protected] Analysis of the tertiary structure of the 23S ribosomal RNA allowed us to determine the order in which different rRNA elements were added to the ribosome as it evolved. The analysis was based on the following suggestions: (1) each new element emerged as a single insertion into the rRNA polynucleotide chain; (2) in each double-helical region, both strands emerged simultaneously as parts of the same element; (3) when a double helix and an adenosine stack formed an A-minor interaction, the element containing the double-helix was considered a more ancient acquisition of the ribosome than the element containing the adenosine stack. Based on these suggestions, we developed an iterative procedure of gradual dismantling the tertiary structure of the ribosomal RNA through removal of those elements that were considered more recent acquisitions of the ribosome. After 59 acts of removal, the remaining element was 220 nucleotide-long and corresponded to the central part of domain V. This element was thus considered the initial one, from which the evolution of the 23S rRNA commenced. All other elements were gradually added to this element as insertions containing all necessary details to dock with the surface of the evolving ribosome without disturbing already existing parts. Bokov, K. and Steinberg, S.V. Nature 457, 977-980 (2009)

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Molecular Mechanisms of Dyskeratosis Congenita Michael Szmyga, U. Thomas Meier Albert Einstein College of Medicine, Department of Anatomy & Structural Biology, 1300 Morris Park Avenue, Bronx, NY 10461 Phone:(718)430-2768 Fax:(718)430-8996 E-Mail: [email protected] Mammalian cells each contain ~150 H/ACA ribonucleoproteins (RNPs) that function in many basic cellular processes including modification of ribosomal RNAs and spliceosomal small nuclear RNAs. Each H/ACA RNP is specified by an H/ACA RNA that associates with four core proteins: NAP57, NOP10, NHP2, and GAR1. This biogenesis also requires at least four assembly factors: SHQ1, NAF1, pontin and reptin. NAP57 is the pseudouridine synthase and is mutated in the predominant X-linked form of the inherited bone marrow failure syndrome dyskeratosis congenita. NAP57 directly interacts with SHQ1, and pathogenic mutations in NAP57 modulate this interaction. SHQ1 release is necessary for H/ACA RNP formation, and occurs through a mechanism not yet understood. Our work consists of three aims: (1) To further study the interaction of NAP57 and SHQ1 in vivo, (2) To understand how SHQ1 is released from NAP57, (3) To define the structural interface between NAP57 and SHQ1. We are currently using an in vivo tethering assay to probe the role of pontin and reptin in SHQ1 release and another in vivo technique, fluorescence fluctuation spectroscopy (FFS) which, for the first time, studies this interaction using proteins intracellularly in their soluble form. Furthermore, through a collaborator, Nick Leulliot, Universite Paris Descartes, Paris, France, we have obtained mutant SHQ1 constructs designed to disrupt the SHQ1:NAP57 interaction based on preliminary x-ray structure information of the complex. Using our in vivo assays we are dissecting the impact of these mutations to verify and define the interaction surface between NAP57 and SHQ1.

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Identifying the pseudoknot structure in the 3’ UTR of Malva Mosaic Virus (MaMV) Rajiv Thapa and Carla A. Theimer The Malva Mosaic Virus is a filamentous virus isolated from common mallow with a genomic organization similar to the group of the Potexvirus genus in the Flexiviridae family. The genomic organization is comprised of a putative RNAdependant RNA polymerase, three overlapping genes coding for the TGB proteins, and finally a coat protein. The MaMV genome consists of a 5’ end capped RNA structure and a 3’ polyadenylated tail. The 3’-UTR is 70 nucleotides long and is predicted to fold into a tRNA-like structure. The polyadenylation signal (AAUAAA) and the conserved hexamer (ACUUAA) sequences in the 3’ UTR are localized in distinct loops of the three stem loop structures in the 3’ UTR. A pseudoknot structure has been proposed to form between Stem Loop 1 (SL1) and Stem Loop 2 (SL2) in the MaMV 3’ UTR. In other closely related potexvirus, a similar pseudoknot has been proposed to form, although the presence of the pseudoknot structure has not been demonstrated. Using UV thermal melting experiments, NMR spectroscopy, and native gel assays, we are investigating the proposed pseudoknot in the virus 3’ UTR structures, using the MaMV sequences as a model system. Mutations have been made to disrupt the pseudoknot structure for biophysical characterization and to provide more information on the structure of the entire 70 nucleotide 3’ UTR.

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A Survey of Known Saccharomyces cerevisiae tRNA Methyltransferases and Human Homolog Candidates William L. Towns and Thomas J. Begley University at Albany, State University of New York, Cancer Research Center, Gen*NY*Sis Center, Room 211, 1 Discovery Drive, Rensselaer, NY 12144-3456. Corresponding author: William Towns. Telephone: 518-591-7163. Fax: 518591-7201. E-mail address: [email protected] Throughout the kingdoms of life tRNA undergoes a number of methyl-based modifications. Although many of the methyl-based modifications are conserved from bacteria to mammals, the function of many of these modifications are still not well understood. The proteins responsible for methylation, tRNA methyltransferases (Trm), have been well characterized in Saccharomyces cerevisiae. In contrast, only a few tRNA methyltransferases have been characterized in Homo sapiens. Using a number of online resources and tools, we have created a database of potential human tRNA methyltransferases, cataloging them by name, accession number, chromosomal location, and measured or theoretical biochemical properties. A total of 33 human proteins matched our search criteria as a Saccharomyces cerevisiae tRNA methyltransferase homolog candidate. Each of the 16 known Saccharomyces cerevisiae tRNA methyltransferases have at least one human homolog, and several (Trm1, 2, 4, 7, 9, 10, 61, and 112) have multiple candidates. While some human candidates have been confirmed as a tRNA methyltransferase, a number of candidates remain unstudied. We believe this database can serve as a starting point for future analysis of human tRNA methylation behavior.

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Molecular Mechanics Analysis of Minimum Energy RNA Conformational Dynamics Pathways Keith P. Van Nostrand1, Scott D. Kennedy1, Douglas H. Turner2, David H. Mathews1* 1. Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave. Box 712, Rochester, NY 14642 2. Department of Chemistry, University of Rochester *Tel. (585)-275-1734, Fax (585)-275-6007, [email protected] Conformational changes are important in RNA for both binding and catalysis. Computational methods were developed for exploring and understanding pathways for defined conformational changes. The conformational change system investigated was a non-canonical pair. In an NMR structure of an AA mismatch in the sequence: 5’ GGUGAAGGCU3’ 3’PCCGAAGCCG 5’ (P = purine), the AA non-canonical pair was in conformational exchange between a minor and major conformation. The conversion of the major trans Hoogsteensugar to the minor trans sugar-Hoogsteen non-canonical pair occurs on the order of tens of microseconds. The AMBER molecular mechanics software package was used to model conformational change pathways. Nudged Elastic Band (NEB) was used to predict minimal potential energy paths for a series of all-atom images of the system along the path. Predicted pathways from NEB were analyzed and a reaction coordinate determined for the conformational change. Umbrella sampling was then used to predict the free energy profile. Umbrella sampling was done using 25 windows of 10 degrees each with 12 ns of sampling per window for 6 different random number seeds. Total sampling involved 1.8 microseconds of MD spanning about 2 years of CPU time. A reversal in relative stability of the major and minor structure indicated by the free energy profile suggests improvement can be made in the AMBER force field.

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An RNA-based Transcription Activator Stimulates Transcription by a Mechanism of Molecular By-pass Shengchun Wang Department of Biological Sciences and Institute for RNA Science and Technology University at Albany, State University of New York, Albany, NY 12222, USA 518-591-8858, [email protected] RNA aptamers have been generated through in vitro selection and extensively used to modulate the activity of their targets, mostly proteins. Most intracellularly delivered RNA aptmers behave as protein inhibitors, much like traditional small molecule drugs. On the other hand, there are some RNAs that stimulate certain intracellular process, although the mechanism behind their stimulatory activity is not clearly elucidated. We sought to expand the utility of RNA aptamers through a rational molecular procedure, in which multiple RNA aptamers were incorporated into a composite construct. This resulting molecule possesses multiple sites recognizable by various aptamer targets, thus creating novel connection among target molecules to realize designated functions. In this example, a site specific RNA aptamer targeting a general transcription factor TFIIB was designed to be the activation domain of an RNA-based transcription activator. With the help of several existing components in the yeast three-hybrid system, this transcription activator RNA (taRNA) is able to stimulate transcription of reporter gene to a level comparable to known protein activators. We propose that the activity of taRNA has the potential to be strictly regulated, in which a regulatory domain of taRNA is added and is separated from its functional domain. The resulting composite RNA is subject to allosteric control that activity of taRNA is either enhanced or inhibited by altering the conformation of regulatory domain.

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U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP Guowei Wu, Mu Xiao, Chunxing Yang, and Yi-Tao Yu Department of Biochemistry and Biophysics, University of Rochester Med Ctr Corresponding Author: Yi-Tao Yu, Tel: 585-275-1271, Fax: 585-275-6007 [email protected] All pseudouridines identified in RNA are considered constitutive modifications. Here, we demonstrate that pseudouridylation of S. cerevisiae U2 snRNA can be conditionally induced. While only three known pseudouridines ( 35, 42 and 44) are detected in U2 under normal conditions (log phase), nutrient deprivation (stationary phase) leads to additional pseudouridylation at positions 56 and 93. Pseudouridylation at position 56 can also be induced by heat shock. Detailed analyses have shown that Pus7p, a single polypeptide pseudouridylase known to modify U2 at position 35 and tRNA at position 13, catalyzes 56 formation, and that snR81 RNP, a box H/ACA RNP known to modify U2 snRNA at position 42 and 25S rRNA at position 1051, is responsible for 93 formation. Using mutagenesis, we have further demonstrated that the inducibility is attributed to the imperfect consensus sequences of the substrates. Our results thus demonstrate for the first time that pseudouridylation of an RNA can be induced at sites with imperfect sequences, and that Pus7p and snR81 RNP can catalyze U2 pseudouridylation at both constitutive and inducible sites.

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Multilign: An Algorithm to Predict Secondary Structures Conserved in Multiple RNA Sequences Zhenjiang Xu1 and David H. Mathews1,2 1

Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, US

2

Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY, US 2

Corresponding author: [email protected] With discovery of diverse roles for RNA, its centrality in cellular functions has become increasingly apparent. Structure prediction is an attractive tool to study RNA function-structure relationship with high speed and low cost. A new algorithm, called Multilign, is presented to find the lowest free energy RNA secondary structure common to multiple sequences. Multilign is based on Dynalign, which is a program that simultaneously aligns and folds two sequences to find the lowest free energy conserved structure. For Multilign, Dynalign is used to progressively construct a conserved structure from multiple pair-wise calculations, with one sequence used in all pair-wise calculations. A base pair is predicted only if it is contained in the set of low free energy structures predicted by all Dynalign calculations. In this way, Multilign is able to improve prediction accuracy by keeping the genuine base pairs and excluding competing false base pairs. Multilign predicts secondary structures of multiple sequences with computation complexity linear in the number of sequences. Multilign was tested on extensive datasets of tRNA, 5S rRNA, Signal Recognition Particle RNA, RNase P RNA, and small subunit rRNA and its prediction accuracy is among the best of available algorithms. The results show Multilign can run on long sequences (>1,500 nt) and an arbitrarily large number of sequences. The algorithm is implemented in ANSI C++ and will be available as part of the RNAstructure package at: http://rna.urmc.rochester.edu.

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Structural Basis For Histone mRNA Recognition By Phosphorylated SLBP Minyou Zhang and Roopa Thapar Department of Structural and Computational Biology, SUNY at Buffalo, and Hauptman-Woodward Institute, 700 Ellicott Street, Buffalo, NY 14203. E-mail: [email protected] Histone mRNAs end in a conserved 26 nucleotide hairpin that binds StemLoop Binding Protein (SLBP). Phosphorylation of the SLBP RNA binding and processing domain (RPD) at a conserved threonine in the sequence HPKTPNK is important for histone mRNA binding and processing. We will report on the effect of threonine phosphorylation of the SLBP RPD on the structure, dynamics, and kinetics of interaction with histone mRNA. Solution NMR Studies show that the SLBP RPD is an all α-helical domain that is structurally unstable in the absence of RNA. The SLBP RPD undergoes phosphorylation-directed prolyl isomerization and exchanges between multiple conformers at physiological pH. Phosphorylation of the SLBP RPD acts as a molecular switch to promote stabilizing interactions with the RNA in the SLBP-RNA complex. The protein undergoes a disorder-to-order transition when it binds RNA. Our studies provide new structural insights as to how RNA-binding proteins may be regulated by reversible posttranslational modifications such as phosphorylation and are broadly applicable to other RNA-binding proteins.

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The Study of tRNA Modifications by Molecular Dynamics XiaoJu Zhang and David Mathews* Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave. Box 712, Rochester, NY 14642 *Tel. (585)-275-1734, Fax (585)-275-6007, [email protected] Modified nucleosides are prevalently found in tRNA [1]. Experimental studies revealed that modifications play an important role in tuning tRNA activity [1, 2]. Molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA structure and dynamics. The X-ray crystal structures of tRNA-Asp [3], tRNA-Phe [4], and tRNA-iMet [5], both with and without modifications, were used as initial structures for 100 ns time scale MD trajectories with AMBER [6]. Force field parameters were built using the procedure of Cornell et al. [7] for 17 nonstandard tRNA residues, including three 5’-terminal phosphate nucleotides. Three independent tRNA trajectory calculations with different random number seeds were performed simultaneously. The tRNA-iMet trajectories showed that N6-threonyl-carbamoyladenosine at position 37 stabilized C34, preventing it from flipping out of the anticodon loop and contacting the anticodon with its long side chain. The global RMSDs showed that all the simulations were stable. Regional RMSDs of anticodon stem-loop displayed that modified tRNA-Asp and tRNA-Phe had lower RMSDs than the corresponding unmodified tRNAs; however, modified tRNA-iMet was slightly higher than the unmodified tRNA-iMet, which was likely caused by fluctuations of the long side chain of the modification at position 37. 1. 2. 3. 4. 5. 6. 7.

Annu Rev Biochem, 1987. 56: p. 263. Mol Microbiol, 1993. 8(6): p. 1011. Acta Crystallogr A, 1988. 44 ( Pt 2): p. 112. Biophys J, 2000. 79(5): p. 2276. EMBO J, 1991. 10(10): p. 3105. J Comput Chem, 2005. 26(16): p. 1668. J Comput Chem, 1995. 16(11): p. 1357-1377.

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