Biochemical research on oogenesis. Binding of tRNA to the [PDF]

In previtellogenic oocytes of Xenopus laevis, nearly all tRNA is included in nucleoprotein particles (thesau- risomes) s

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THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, h e .

Vol. 262, No. 2, Isaue of January 15, pp. 654-659.1987 Printed in U S A .

Biochemical Research on Oogenesis BINDING OF tRNA TO THE NUCLEOPROTEINPARTICLES PREVITELLOGENIC OOCYTES*

OF XENOPUS LAEVIS

(Received for publication, May 8,1986)

Marc le Maire and Herman Denis From the Laboratoire de Bwchimie du Dtvebppement, Centre de Gnitique Molpculuire du Centre Natwnnl de la Recherche Scientifique (Ldoratoire propre du Centre National de la Recherche Scientifque associi a 1’UniuersiteParis VZ),F-91190Cifsur- Yvette, France

In previtellogenic oocytes of Xenopus laevis, nearly menting at more than 100 S.* All tRNAs are represented in all tRNA is included in nucleoprotein particles (thesauthe 42 S particles (9). All of them are fully charged in vivo (9) risomes) sedimentingat 42 S. We evaluate the possi- and somehow protected against deacylation (10). In spite of bility of a tRNA exchange between the particles and this protection, particle tRNA becomes deacylated and rethe ribosomes during protein synthesis. We find that acylated continuously in uiuo, with a turnover rate of a few the particles take up tRNA after a very short incuba- hours (10). Partially purified thesaurisomes can also aminotion i n vitro. In theabsenceofATP,the particles acylate their own tRNA (9, 10) because they are associated preferentially bind charged tRNA. In the presence of with aminoacyl-tRNA synthetases (9). These enzymes are ATP, more tRNA binds to the particles, and the sedi- minor components of the thesaurisomes and clearly distinct mentation coefficient of the integrated tRNA is dis- from the two main particle proteins (9). placed to45 S . When added to nonfractionated homogWe think it unlikely that nearly all tRNA of the cells enates of oocytes togetherwith ATP, poly(U) strongly remains stored in the thesaurisomes for several months, and stimulates the incorporation of radioactive phenylalabecomes charged and discharged many times, without ever nine into tRNA andprotein. The labeled protein (polyleaving the particles or moving from one particle to another. phenylalanine) cosediments with the ribosomes, whereas most of phenylalanyl tRNA cosedimentswith If such a movement does actually occur in uiuo, the particles should be able to take up small amounts of tRNA when the thesaurisomes. These data suggest that the thesaurisomes participate to some extent in proteinsynthesis. incubated in cell-free extracts. In this paper we show that aminoacyl-tRNA does indeed bind to the 42 S particles in TheyreleasechargedtRNA,therebysupplyingthe vitro. We also study the influence of several factors (energy, ribosomes with activated aminoacids.Discharged tRNA is then taken up, reacylated, and stored in the aminoacylation state of tRNA, and syntheticpolyribonucleoparticles until the next round of peptide bond forma- tides) on the integration of tRNA into the particles. We find tion.Theaminoacylationandstoragefunctions are that addition of poly(U) to cell-free extracts of oocytes probably carried out bytwo very unequal populations strongly stimulates the incorporation of phenylalanine into of particles. The main subclass of particles (42 S ) binds protein by the ribosomes and the turnover of phenylalanyland stores tRNA in an ATP-independent manner. A tRNA in the 42 S particles. This shows that the ribosomes much smaller subclass of particles (45 S) is responsible can recruit tRNA from the 42 S particles in uitro and suggests for reacylation of discharged tRNA. that discharged RNA released from the ribosomes is taken up and reacylated by the thesaurisomes. Our results also indicate that two very unequal populations of particles participate in the exchange of tRNA with the cytosol and with the riboThe protein-synthesizing system of Xenopus iueuis previ- somes. tellogenic oocytes has several unusual properties. These cells EXPERIMENTAL PROCEDURES contain few ribosomes (1, 2), but actively synthesize protein (3). The most abundant RNA species in small oocytes are not Materials-Labeled amino acids and uridine were purchased from 28 and 18 S RNA, but 5 S RNA and tRNA ( 4 5 ) . Only a very CEA, Saclay, France. Poly(A) and poly(U) were from Boehringer small fraction of the total tRNA content of the oocyte is free Mannheim. Poly(C) was from Serva andpoly(C,U,G) was from Miles. in thecytosol. More than 90% of the cell’s tRNA isassociated Previtellogenic oocytes were obtained from whole ovaries of X.laevis females and handled as described in our previous publicawith nucleoprotein particles (thesaurisomes) sedimenting at immature tions (1, 6,9, 10). 42 S (1).These particles are made up of four subunits, each Purification and Labeling of tRNA-tRNA was extracted from of which contains tRNA, 5 S RNA, a M,50,000 protein, and mature ovaries by the standard dodecyl sulfate-phenol method and a M, 40,000 protein in the following molar ratios: 3:1:21 purified by filtration on a column of Sephadex G-100 (4). During all (Refs. 1,5-8). A small fraction of total tRNAis also associated purification steps, the pH of the RNA samples was kept below 5 so with messenger ribonucleoprotein particles (mRNPs)’ sedi- that the full acylation state of tRNA was preserved (9).When *This paper is No. 19 of the present series. Paper No. 18 is by Denis and le Maire(10).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviation used is: mRNP, messenger ribonucleoprotein particle.

necessary, tRNA was discharged in 1 M NH4HC03, pH 9,for 1 h at room temperature. tRNA was acylated by means of liver aminoacyltRNA synthetases (9),either with one or with several labeled amino acids. tRNA was labeled in its nucleic acid moiety by incubating cultured cells with [3H]uridine (10). Binding of tRNA to the Particles-The ovaries were homogenized in 3-5 volumes of 50 mM Tris/HCl, pH 7.6,25 mM KCl, 5 mM MgCl,.

654

* M. le Maire and H. Denis, unpublished observation.

655

Biochemical Research on Oogenesis

,210

+ATP

-

150

I-

-200

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4

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8

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TUBENUMBER

8 12 TUBENUMBER

16

FIG. 1. Binding of [86S]methionyl-tRNAto the 42 S particles in the presence of nonlabeled methionine (M and )acylation of particle tRNA with [S6S]methionine during incubation with a homogenate of previtellogenic oocytes (W). One immature ovary was homogenized in 750 pl of 50 mM Tris/ HC1, pH 7.6,25 mM KCl, 5 mM MgC12. The homogenate was clarified by a low-speed centrifugation (10,000 rpm for 10 min) and divided into two equal parts. One part was incubated at 30 'C for 15 min with 5 pgof methionyl-tRNA (640,000 counts/min/pg) in 50 mM Tris/HCl, pH 7.6,25 mMKC1, 5 mM MgC12, 10 mM ATP, 5 mM phospho(enol)pyruvate, 20 pg/ml pyruvate kinase, 0.2 mM L-methionine. The other partwas incubated under the same conditions with 5 pg of nonlabeled tRNA and 1.4 pmol of [36S]methionine(3 X lo6 counts/min). Both samples were cooled,loaded on 15-30% sucrose density gradients made up in the homogenization buffer, and spun at 40,000 rpm for 4 h in a SW 41 rotor. The graph was obtained by superposing the radioactivity profiles given by the two samples. The absorbance profile is from the sample incubated with [%S]methionyl-tRNA. The 42 S particles in each assay contained approximately 50 pg of tRNA. Note that in the first experiment (M the ), 42 S and 4-7 S peaks contain 10.5 and 0.9%, respectively, of the total amount of radioactivity added to the ovary homogenate. Volume of the fractions was 0.6 ml. FIG. 2. Binding of aminoacyl-tRNA to the 42 S particles in the presence(o"0) and in the absence (M of) ATP. One pair of ovaries was homogenized in 1ml of buffer and centrifuged as described in Fig. 1. The homogenate was divided into two equal parts, mixed with 36 pg of tRNA labeled with all amino acids except cysteine (6,000 counts/min/pg), and incubated at 30 "C for 10 min in 50 mM Tris/HCl, pH 7.6,25 mM KCl, 10 mM MgCl,, 0 or 1mM ATP, 0 or 1mM phospho(enol)pyruvate, 0 or 4 pg/ml pyruvate kinase. Both samples were cooled, loaded on 1530% sucrose density gradients overlying a cushion of 66% sucrose, and spun at 32,000 rpm for 14 h. The absorbance profile is from the sample incubated without ATP. The 42 S particles in each assay contained approximately 150 pg of tRNA. Volume of the fractions was 0.6 ml. FIG. 3. Binding of [36S]methionyl-tRNA to purified 42 S particles in the presence (M and )in the absence (U of) ATP. Two pairs of immature ovaries were homogenized and fractionated by sucrose density centrifugation, as described in Fig. 1. The particle-containing fractions were pooled and divided into two 320-p1 aliquots. Each aliquot was incubated at 30 "C for 10 min with 20 pg of [36S]methionyl-tRNA(30,000 counts/ min/pg) in 50 mM Tris/HCl, pH 7.6,25 mM KCl, 5 mM MgCl,, 22% sucrose, 0 or 10 mM ATP. Both samples were cooled, diluted by adding 600 pl of the homogenization buffer, loaded on 1 5 3 0 % sucrose density gradients, and spun again at 32,000 rpm for 12 h. The absorbance profile is from the sample incubated without ATP. The 42 S particles in each assay contained approximately 30 pg of tRNA. Volume of the fractions was 0.6 ml. The supernatantwas clarified by centrifugation a t 12,500 X g (10,000 rpm). Aliquots of the supernatant were incubated at 30 "C for 5-30 min with labeled tRNA in the presence or in the absence of ATP, GTP, UTP, CTP, and synthetic polyribonucleotides. The samples were cooled to 0 "C and loaded on 15-30% sucrose density gradients made up in the homogenization buffer. The gradients were spun for 4-5 h at 40,000 rpm or overnight at 22,000 or 32,000 rpm in a SW 41 rotor. Each fraction of the gradient was precipitated with 12.5% trichloroacetic acid, filtered on WhatmanGF/C fiber glass discs, and counted in 5 ml of scintillation fluid. In some experiments, incubation with labeled tRNA was carried out with purified 42 S particles. In this case, the sample was diluted at the end of the incubation in order to lower its density and centrifuged again at 32,000 rpm for 12-14 h. Incorporation of Labeled Amino Acids into tRNA and ProteinWhole ovary homogenates were incubated at 30 "C for 1h with labeled amino acids. The incubation mixture (100-500 p l ) contained 50 mM Tris/HCl, pH 7.6,80-100 mM KC1,lO mM MgC12,3-5 mM ATP, 3-5 mM phospho(eno1)pyruvate. 10-40 pg/ml pyruvate kinase, 10-50 pCi/ ml labeled amino acid, and various amounts of synthetic polyribon-

ucleotides. The amount of radioactivity incorporated into high-molecular mass material was measured directly or after fractionationof the cell-free extracts by sucrose density centrifugation. The ovary homogenates or the fractions of the sucrose gradients were divided into two sets of 25-100 pl aliquots. The first set was brought to pH5 by adding 0.1 volume of 1 M sodium acetate buffer. The second set was digested for 45 min at 37 "C with 100 pg/ml pancreatic ribonuclease in 10 mM EDTA, pH 7.6. All aliquots were transferred to 1X 1-cm sheets of Whatman No. 3" paper. After drying, the filters were dipped for 20 min in ice-cold 10% trichloroacetic acid, rinsed four times in 3% trichloroacetic acid, twice in 95% ethanol, dried again, and counted in 2.5 ml of scintillation fluid. The amount of radioactivity incorporated into tRNA is given by the difference between the radioactivity in the pH5 samples and theradioactivity in the ribonuclease-treated samples. RESULTS

Binding of Arninoacyl-tRNA to the 42 S Particles-When homogenates of previtellogenic oocytes are incubated with

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Biochemical Research on Oogenesis

[35S]methionyltRNA and analyzed by sucrose density centrifugation, a fraction of the added tRNA cosediments with the 42 S particles (Fig. 1). About 99% of the tRNA that was not taken up by the particles became deacylated during incubation with the cell-free extract, centrifugation, and all subsequent operations (Fig. 1). This can be inferred from the almost complete absence of acid-insoluble radioactivity in the 4-7 S region of the sucrose density gradient, which contains the unbound tRNA (Fig. 1). Deacylation of free tRNA during and after the binding reaction cannot be prevented because the conditions which could stabilize the ester bond of aminoacyl-tRNA (low pH) dissociate the 42 S particles? In fact, deacylation of tRNA during the binding reaction and the ensuing purification facilitates the interpretation of the data, since only the aminoacyl-tRNA which cosediments with the particles remains acid-insoluble at theend of the experiment and forms a clear-cut peak (Fig. 1).When no loss of radioactivity can result from deacylation, i.e. when tRNA labeled in its nucleic acid moiety is used for the binding experiments, the amount of tRNA cosedimenting with the 42 S particles cannot be accurately measured (10). The data presented in Fig. 1 can be interpreted to mean that tRNA binds to the 42 S particles in such a way that its ester bond is protected against hydrolysis (10). An alternative explanation would bethat tRNA in the 42 S particles becomes charged with the labeled amino acid released from the added tRNAduring incubation with the ovary homogenates (9). This explanation can be rejected for the following reasons. Even if all the amino acid molecules originally linked to tRNA were released during the incubation, the amount of particle tRNA that could be reacylated would be very small (Fig. 1). Furthermore, reacylation of particle tRNA with the labeled methionine released from aminoacyl-tRNA can be prevented by adding a large excess of cold methionine to theincubation medium. Even under these conditions, a substantial amount of labeled tRNA sediments with the thesaurisomes (Fig. 1). Integration of aminoacyl-tRNA into thethesaurisomes can be observed whether crude ovary homogenates (Figs. 1and 2) or purified particles (Fig. 3) are used in the tRNA-binding experiments, andwhatever precursors are used to label tRNA: uridine (IO), a single amino acid (cysteine, leucine, lysine, phenylalanine, proline, serine, or methionine; Figs. 1 and 3) or a mixture of 19 amino acids (Fig. 2). Addition of ATP to the incubation mixture increases the amount of tRNA which is taken up by the particles (Figs. 2 and 3). ATP also shifts the sedimentation coefficient of the integrated tRNAfrom 42 to 45 S, but does not displace the main particle peak, as revealed by the UV absorbance (Figs. 2 and 3). GTP is less efficient than ATP in stimulating the binding of tRNA. CTP and UTP have little or no effect on this process (data not shown). The results presented in Figs. 1-3 do not depend crucially on the time and on thetemperature of incubation of aminoacyl-tRNA with the ovary homogenates. The amount of tRNA cosedimenting with the particles does not change appreciably if the homogenates are incubated for 5 , 10 or 30 min, at 510 "C, 20-25 "C, or 30 "C.This suggests that the affinity of the 42 S particles for tRNA is very high and that saturation of the binding sites of the particles occurs in the first minutes and perhapsseconds of incubation. The bound tRNA becomes deacylated very slowly (10) and remains associated with the particles during the remainder of the incubation and all subsequentoperations (Figs. 1-3).No indication could be obtained concerning the actual binding rate of tRNA because the binding reaction cannot be rapidly stopped due to the properties of the 42 S particle system. Dilution, acidification,

or alkalinization cause the 42 S particles to dissociate.' Furthermore, the time needed to separate the unbound tRNA from the 42 S particles by any available method (sucrose density centrifugation, gel filtration, electrophoresis) far exceeds the time needed for the binding reaction to occur. Whatever its actual value, the time of the binding reaction appears to be much shorter than the half-life of aminoacyltRNA, which ranges from 30 min to more than 1 h under our experimental conditions (10). A small amount of labeled tRNA moves to the bottom of the tubes during sucrose density centrifugation (Fig. 2). It can be recoveredas a peak of acid-insoluble radioactivity provided a cushion of concentrated sucrose prevents it from forming a pellet on the bottom of the centrifuge tube (Fig. 2). The fastsedimenting tRNA is not associated with the ribosomes or the polysomes, but with the mRNPs.These particles are known to sediment faster than 40 S (11).Deoxycholate, which dissociates the 42 S particles (1) and the mRNPs (12), but not the ribosomes and thepolysomes (12), drastically reduces the amount of labeled tRNA sedimenting at 42 S and more than 100 S (data not shown). The amount of tRNA which is taken up by the thesaurisomes does not increase linearly with the concentration of tRNA in the incubation medium (Fig. 4). The particles become apparently saturatedwith exogenous tRNA, bothin the absence and in the presence of ATP (Fig. 4). This means that the particles can integrate only a limited amount of tRNA. At full saturation, the 42 S particles take up no more than 2 pg of aminoacyl-tRNA/100 pg of endogenous tRNA. In this respect, the behavior of the mRNPsis very similar to thatof the thesaurisomes (Fig. 4). The mRNPs become readily saturated with exogenous tRNA and bind more tRNA in the presence than in the absence of ATP (Fig. 4). Competition Between Charged tRNA and Discharged tRNA for Integration into the 42 S Particles-The 42 S particles take up not only charged tRNA (Figs. 1-4), but also discharged tRNA. This can be demonstrated by using tRNA labeled in its nucleic acid moiety. The 42 S particles take up less dis-

+ATP

FIG. 4. Saturation experimentof an ovary homogenate with aminoacyl-tRNA. Six ovaries were homogenizedin 2.4 ml of buffer and centrifuged as described in Fig. 1. Aliquots of 200 pl from the clarified homogenate were incubated a t 30 "C for 10 min with increasing amounts of [3H]methionyl-tRNA (700 counts/min/pg) in 50 mM Tris/HCl, pH 7.6,25 mM MgCl,, 10 mM MgCl,, 0 or 5 mM ATP. The samples were centrifuged in sucrose density gradients as in Fig. 2, and theamount of acid-insoluble radioactivity sedimenting either at 35-50 S or faster than 80 S (ie. with the mRNPs) was measured. The 42 S particles in each assay contained approximately 100 pg of tRNA.

Research Biochemical charged tRNA than charged tRNA when no ATP is added to the incubation medium (data not shown). The competition experiment shown in Fig. 5 confirms this finding. In the absence of added ATP, charged tRNA inhibits the binding of methionyl-tRNA three to four times more efficiently than does discharged tRNA. When ATP is present in the incubation medium, the difference in competing efficiency between charged and discharged tRNA drops from 3-4 to approximately 1.5 (Fig. 6). In both experiments, discharged tRNA behaves as a competitive inhibitor uersus charged tRNA (Figs. 5 and 6 ) . This means that both forms of tRNA bind to the same site(s) in thethesaurisomes. These sites are not accessible to 5 S RNA, since this molecule does not reduce the binding of aminoacyl-tRNA to the 42 S particles (data not shown). Influence of Synthetic Polyribonucleotideson the 42 S Particles-So far, we have shown that the42 S particles can take up small amounts of tRNA (Figs. 1-6). Is this a one-way process or do the particles exchange tRNA with other cell components and especially with the ribosomes? To answer this question, we stimulated protein synthesis with polynucleotides in cell-free extracts of oocytes, which contain both

on Oogenesis

657

2.5

IO

LABELED

3.o

t RNA / NON-LABELED

tRNA

FIG. 6. Competitionexperimentbetween [85S]methionyltRNA and nonlabeled tRNA for binding to the 42 S particles in the presence of5 mM ATP. Four ovaries were homogenized and incubated as described in Fig. 5 with 1.2 pg of [36S]methionyl-tRNA (100,000 counts/min/pg) and increasing amounts (16-130 pg) of nonlabeled tRNA.

-

-

thesaurisomes and ribosomes (1, 5 ) . When added to such homogenates together with ATP, poly(U) increases 3-30-fold the incorporation of [3H]phenylalanine into tRNA and 10200-fold the incorporation into protein (datanot shown). ? r \ Poly(U) has little or no influence on the incorporation of other amino acids. Poly(A) slightly stimulates (2-5-fold) the 2.o incorporation of [3H]lysineinto tRNA and protein of cell-free extracts. In contrast to poly(U) and poly(A), poly(C) has no charged t R N A distinct influence on the incorporation of the amino acid \ / specified by the codons it contains, i.e. proline. Fractionation of the cell-free extracts by sucrose density centrifugation 1.5 shows that stimulation of protein synthesisby poly(U) occurs in the ribosome-containing fractions (80 S), whereas stimulation of tRNA aminoacylation occurs in the particle-containing fractions (30-40 S; Fig. 7). I The increase inturnoverrate of particle phenylalanyl11.0" ( 0.1 0 tRNA does not occur in the absence of ribosomes, since LABELED t RNA /NON-LABELED tRNA poly(U) only moderately stimulates the incorporation of [3H] FIG.5. Competitionexperimentbetween[s5S]methionylphenylalanine into tRNAby purified 42 S particles.' FurthertRNA and nonlabeled tRNA for binding to the 42 S particles in the absence of added ATP. Two ovaries were homogenizedin 2 more, poly(U) and other synthetic polyribonucleotides have ml of buffer and centrifuged as described in Fig. 1. Aliquots of 225 pl little direct influence on the binding of aminoacyl-tRNA to from the clarified supernatant were incubated at 22 "C for 15 min the 42 S particles (Table I). Poly(U) slightly stimulates, but with 4 pg of [35S]methionyl-tRNA (164,000 counts/min/pg) and in- poly(C) and poly(C,U,G) inhibit the integration of all amicreasing amounts (24-180 pg) of nonlabeled tRNA. The amount of acid-insoluble radioactivity sedimenting with the 42 S particles inthe noacyl-tRNAs that have been tried (Table I). The influence absence of competitor is taken as 1, and the fraction of the radioac- of poly(A) on the 42 S particles is peculiar, since it selectively tivity recovered in the presence of competitor isnoted as Y. The data increases the amount of lysyl-tRNA which binds to the 42 S are presented as a double-reciprocal plot of 1 - Y uersus the mass particles (Table I). This stimulation occurs only in the presratio of labeled tRNA tononlabeled tRNA in theincubation mixture. ence of ATP (data not shown). 2.5

-

/

/

A value of 2 on the ordinate scale means that the amount of labeled tRNA bound to the particles has been reduced by half ( Y = 0.5 and 1/1 - Y = 2). This value should be obtained after adding 4 pgof nonlabeled aminoacyl-tRNA to theincubation mixture (position 1 on the abscissa scale,outside the frame). In fact, the 50% inhibition point by charged tRNA is reached at a smaller value on the abscissa scale (0.25, corresponding to a mass ratio of nonlabeled tRNA to labeled tRNA of 4). This displacement is due to thefact that at theconcentrations of tRNA used, the 42 S particles are not saturated with exogenous tRNA (see Fig. 4).

DISCUSSION

The nucleoprotein particles presentin homogenates of previtellogenic oocytes take up small amounts of aminoacyltRNA after a very short time of incubation (Figs. 1-3). The bound tRNA shares one important property with the endogenous tRNA of the 42 S particles: its ester bond is protected against hydrolysis. This suggests that the labeled tRNA co-

658

Biochemical Research on Oogenesis

FIG. 7. Influence of poly(U)on the incorporation of [SH]phenylalanine into tRNA and protein in an ovary homogenate. Aclarifiedhomogenate fromtwoovarieswasdivided into two 375-pI aliquots. The first aliquot wasincubated at 30 "C for 1 h in 50 mM Tris/ HCI, pH 7.6, 82 mM KCl, 10 mM MgCI,, 3 mM ATP, 5 mM phospho(eno1)pyruvate, 20p g / m l pyruvate kinase, and 32 &i/ml [3H]phenylalanine (25 Ci/ mmol). The other aliquot was incubated in the same medium, butcontaining 600 pg/ml poly(U). At the end of the incubation period, L-phenylalanine (final concentration 1 mM) was added to both aliquots. The samples were cooled, loaded on two15-30%sucrosedensity gradients,andspun at 40,000 rpmfor 225 min in a SW 50 rotor. The amount of radioactivity incorporated into tRNA and protein was measured in each fraction. Note that the incubation conditions used in this experiment partially dissociated the 42 S particles (whichare normally tetramers)into trimers (34 S) and evendimers(25 S) ( 6 ) .Volume of the fractions was 0.2 ml.

,-

0

TUBE NUMBER

TABLE I Influence of synthetic polyribonucleotides on the binding of amimacyl-tRNA to the 42 S particles in the presence of 5 mM ATP

".

would range from 0.1 to 0.5 mM (2.5-12.5 mg/ml) (4, 13). Such a high concentration could be incompatible with efficientproteinsynthesis because of undesired tRNA-tRNA Polyribonucleotide" interactions, due to association between complementary anAmino acid usedto label tRNA ticodons (14, 15). poly(A) PO~Y(C) poly(U) poly(C,U,G) In addition to storage, the thesaurisomes fulfill another 0.8 0.7 1.65 0.7 [3H]leucine function, i.e. reacylation of discharged tRNA released from 0.6 1.75 0.6 1.5-2.5 [3H]lysine the ribosomes. We suggest that the thesaurisomes and the 1.05 0.6 1.4 0.7 [%]methionine 0.7 1.75 0.7 0.95 mRNPs take up exogenous tRNA (Figs. 1-3) because they 13H]phenylalanine 0.6 1.6 0.6 0.7 f'Hjprohne have empty tRNA-binding sites. We further assume that 0.7 1.55 0.7 All except cysteine 0.85 storage and aminoacylation of tRNA are carried out by two 'In each experiment, 3-15 concentrations of polyribonucleotide different subclasses of particles. The bulk of the particles were used. The data presented correspondto the maximum effect of which is responsible for the main absorbance peak (42 S ) each polyribonucleotide.The amount of aminoacyl-tRNA bound to binds charged tRNA in preference to discharged tRNA (Fig. the particles inthe absence of added polyribonucleotideis set as 1. 5) whether or not ATP is present. In this way, discharged tRNA fails to be taken upby the 42 S particles and remains sedimenting with the 42 S particles and with the mRNPs is in thecytosol. A smaller subclass of particles which sediments integrated in these complexes in such a way that its 3'-end at 45 S, i.e. slightly faster than thebulk of the thesaurisomes, becomes inaccessible to water (10).ATP stimulates the bind- takes up tRNA only when ATP is present (Figs. 2 and 3). ing of tRNA not only to the thesaurisomes but also to the These particles bind discharged tRNA in preference to mRNPs (Figs, 2 and 4). Both kinds of complexes probably charged tRNA, since addition of ATP reduces the preferential contain similar tRNA-binding sites. These sites aresensitive to deoxycholate and nearly insensitive to synthetic polyribo- affinity of the particles for charged tRNA (Fig. 6). We connucleotides (Table I). Only lysyl-tRNA binds to the thesau- sider the 45 S particles as a metabolically active subclass of risomes in a template-dependent manner (Table I), for some thesaurisomes and the 42 S particles as storage places for reason that we cannot explain. However, the stimulation of aminoacyl-tRNA. The 45 S particles also have empty tRNAlysyl-tRNA binding caused by poly(A) is not very strong binding sites andcarry out tRNA aminoacylation in vivo and (Table I). The stimulation caused by poly(U) is not specific, in vitro because they are associated with one or several amisince the binding of phenylalanyl-tRNA is not selectively noacyl-tRNA synthetases (9). The model outlined above holds that the45 S particles are increased by this polyribonucleotide (Table I). the main aminoacylation center in previtellogenic oocytes. The experiments described in this paper and in previous Little aminoacylation indeed occurs outside the thesauripublications (1,9, 10) allow us to propose a dual function for the thesaurisomes, i.e. storage of small RNA molecules (1, 5) somes in cell-free extracts of immature ovaries (9). This and reacylation of tRNA. The 42 S particles store 5 S RNA suggests that reacylation of discharged tRNA in the oocytes and tRNA in such a way that these molecules are protected is carried out not by free, but by particle-associated aminoagainst degradation (7) and deacylation (10).The storage acyl-tRNA synthetases. Several questions arise concerning function is beneficial for anotherreason: it enables the oocyte the structure of the 45 S particles. Are these particles perto keep its concentration of free tRNA to a low value. If all manently or transiently associated with the aminoacyl-tRNA tRNA were released from the 42 S particles into the cytosol, synthetases? In the former case, the 45 S particles would the totalconcentration of free tRNA inprevitellogenic oocytes correspond to a fixed subclass of thesaurisomes; discharged

Biochemical Research on Oogenesis tRNA would be reacylated in the 45 S particles and transferred to the 42 S ones either directly or via the cytosol. In the latter case, all the 42 S particles would in turn be associated with the aminoacyl-tRNA synthetases. After completion of the aminoacylation reaction, charged tRNA would remain bound to theparticles, but theenzymes would dissociate from them, thus causing their sedimentation coefficient to shift from 45 to 42 S. Another point which is not settled concerns the differential affinity of the particles for charged and discharged tRNA. We interpretourdata (Figs. 5 and 6) by assuming that in the presence of ATP the 45 S particles preferentially bind discharged tRNA. This could be due to a conformation change triggered by ATP. Another possibility wouldbe that the aminoacyl-tRNA synthetases associated with the particles are responsible for the observed binding preference. In the presence of ATPand amino acids, an aminoacyl adenylate is formed on the active site of the aminoacyl-tRNA synthetases. Under these conditions, the enzymes can be expected to preferentially bind discharged tRNA. After reacylation, tRNA would be transferred to the particles (10). The model of tRNA uptake outlined above explains two observations concerning the metabolic activity of the thesaurisomes. First, poly(U) stimulates the turnover of phenylalanyl-tRNA in the thesaurisomes when added to nonfradionated ovary homogenates (Fig. 7). This accelerated turnover can be ascribed to anincreased recruitment of phenylalanyltRNA by the ribosomes. Discharged tRNA is then released into thewater phase, taken up, and reacylated by the thesaurisomes. Second, a strong aminoacylation activity is associated with the 45 S particles purified by sucrose density centrif~gation.~ Theseparticlescan aminoacylate their own tRNA when incubated with ATP. We interpret thisproperty of the thesaurisomes in the following way. When incubated in uitro, the particles release some of the tRNA they contain. This is due to an equilibrium shift caused by dilution. Free tRNA is no longer protected against deacylation (10). After being discharged in the water phase, it is takenupand reacylated by the particles. In thisway, a futilecycle becomes established assoon as theparticles areincubated in vitro with ATP (9). The cycle is driven by the opposite affinities of the two particle subclasses for charged and discharged tRNA. The rate-limiting step(s) in this cycle could be the amount of M. le Maire and H. Denis, manuscript in preparation.

659

aminoacyl-tRNA synthetases associated with the particles (9) and/or the availability of discharged tRNA, which in turn depends on thehalf-life of the individual aminoacyl-tRNAs. The experiments described in this paper and in previous publications (9,lO) cannot be interpreted without postulating a steady exchange of tRNA between the ribosomes, the cytosol, and the thesaurisomes. No such movement can be detected as far as5 S RNA i s concerned. Newly made 5 S RNA becomes distributed between the ribosomes and the thesaurisomes and remains apparently confined to its site of integration until the end of the previtellogenic period (7, 16). In somatic cells which apparently lack storage particles (5), the tRNA cycle is simpler than in the oocytes and presumably restricted to a shuttlebetween the ribosomes and thecytosol, eucaryotic elongation factor Tu acting as a carrier in this cycle to deliver charged tRNA to the proper site of the ribosome (17). The role of the latter factor in the alleged exchange of tRNA between the ribosomes and the thesaurisomes remains to be elucidated. We do not exclude the possibility that some kind of association might exist between eucaryotic elongation factor Tu and the 42 S particles. Acknowledgment-We thank Dr. C. Monteilhet for critically reading the manuscript of this paper. REFERENCES 1. Denis, H., and Mairy, M. (1972)Eur. J. Biochem. 26,524-534 2. Dixon, L.K.,and Ford, P. J. (1982)Dev. Biol. 91,474477 3. Dixon, L. K.,and Ford, P. J. (1982)Dev. Bwl. 93,478-497 4. Mairy, M., andDenis, H. (1971)Deu. Biol. 24, 143-165 5. Ford, P. J. (1971)Nature 233, 561-564 6. Picard, B., le Maire, M., Wegnez, M., and Denis, H. (1980)Eur. J. Bwchem. 109,359-368 7. Denis, H., and le Maire, M. (1983)SubceU. Biochem. 9, 263-297 8. Kloetzel, P., Whitfield, W., and Sommerville, J. (1981)Nuleic Acids Res. 9,605-621 9. Wegnez, M., and Denis, H. (1979)Eur. J. Biochem. 98,67-75 10. Denis, H., and le Maire, M. (1985)Eur. J. Biochem. 149, 549556 11. Darnbrough, C. H., and Ford, P.J. (1981)Eur. J. Biochem. 113, 415-424 12. Darnbrough, C.,and Ford, P. J. (1976)Deu. Biol. 60,285-301 13. Taylor, M. A., and Smith, D. (1985)Dev. Bwl. 110,230-237 14. Grosjean, H., Soll, D. G., and Crothers, D. M.(1976)J. Mol. Biol. 103,499-519 15. Grosjean, H., de Henau, S., and Crothers, D.M. (1978)Proc. Natl. Acad. Sei. U. S. A. 76, 610-614 16. Mairy, M., and Denis, H. (1972)Eur. J. Bwchem. 25,535-543 17. Haselkom, R., and Rothman-Denes, L. B. (1973)Annu. Rev. Biochem. 42,397-438

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