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Termination of Quiescence in Crustacea

Termination of Quiescence in Crustacea THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 46, Issue of November 14, pp. 28912–28917, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. THE ROLE OF TRANSFER RNA AMINOACYLATION IN THE BRINE SHRIMP ARTEMIA* (Received for publication, July 31, 1997) Margreet Brandsma, George M. C. Janssen, and Wim Mo ¨ ller‡ From the Department of Medical Biochemistry, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands After 30 years of research on the re-activation of translation In quiescent embryos of the brine shrimp Artemia, the level of aminoacylation of transfer RNAs is low. in Artemia, it is clear that quiescent and developing embryos During resumption of development the charging level contain approximately equal amounts of ribosomes (8), mRNA of transfer RNAs increases, concomitant with the acti- (9), initiation factors (10), elongation factors (11), and termina- vation of protein synthesis. The total level of charging tion factors (12), which are all equally active in in vitro trans- rises dramatically from an average of 4% to 50% within lation assays. At the onset of development, a slow but definite a period of 24 h of development. The restriction of in shift from 80 S ribosomes to polysomes can be observed (5, 13). vitro translation of the quiescent embryo extract can However, extracts from quiescent embryos are inactive in be partially released by the addition of charged ami- translation of their endogenous mRNAs (14). This paradox has noacyl-tRNA, which apparently starts the flow of ribo- led several investigators to search for specific inhibitors and somes into polyribosome structures. Complete reacti- activators of translation in extracts from non-developing and vation of translation by aminoacyl-tRNA occurs when developing embryos, respectively (15). Until now, none of these mRNA from preformed mRNA-ribosome complexes, approaches has provided a definite answer. like the polyribosomes extracted from developing em- Since ribosomes, factors, and mRNAs appear to be normal in bryos or poly(U)-programmed ribosomes, are offered the quiescent embryo, we have explored the level of charged to quiescent embryo extracts. With respect to the tRNA during development. Although the amount of tRNA as mechanism of in vivo recharging of tRNAs, we ob- such does not change significantly during development (16), served that the level of several aminoacyl-tRNA syn- the degree of tRNA aminoacylation has until now received little thetases increase during development. Methionyl- attention. The results of such studies are presented here. tRNA synthetase rises more than 10-fold. In the case of valyl-tRNA synthetase, the activation is lower and EXPERIMENTAL PROCEDURES shown to be due to the de novo synthesis of its mRNA Culturing Conditions—Dried Artemia embryos (San Francisco Bay and the corresponding protein product as well. We Brand Inc., San Francisco, CA) were washed with 2% NaOCl as de- conclude that protein synthesis and thereby the grad- scribed (17) and either used directly (quiescent embryos) or cultured for ual animation of cryptobiotic Artemia embryos is de- the time indicated in aerated artificial sea water at 27 °C (developing termined to a large extent by the rate by which ami- embryos). Under these conditions, approximately 80% of the embryos noacyl-tRNAs are replenished during development at emerge from their shells within 20 h. both the initiation and elongation level. In Vivo tRNA Charging Levels—Dried embryos (25 g) were cul- tured for the time indicated and washed with distilled water and 50 mM NaAc, pH 4.5, 150 mM NaCl. The volume was adjusted to 75 ml with the same buffer, 50 ml of phenol (saturated with the same Multicellular organisms like the crustacean Artemia can sur- buffer) was added, and this mixture was homogenized three times for vive long periods of environmental stress by entering a crypto- 45 s with a Polytron homogenizer. Upon centrifugation for 12 min at biotic stage (see Refs. 1– 4 for reviews). Tissue culture cells can 3000 3 g in a Beckman JA 10 rotor, the aqueous (upper) phase was protect themselves against adverse growing conditions by en- extracted two more times with the same amount of acidic phenol. tering the G phase of the cell cycle. In both cases, the rate of RNA from the final upper phase was precipitated with ethanol (18). The dried pellet was dissolved in 15 ml of 10 mM NaAc, pH 4.5, and protein synthesis is reduced to a low level and its reactivation lithium chloride was added to a concentration of 0.8 M, followed by is a prerequisite for the cells to re-enter the cell cycle (5, 6). For centrifugation for 20 min at 9000 3 g in a Sorvall SS 34 rotor. The a better understanding of the processes that determine cell tRNA present in the supernatant was reprecipitated with ethanol growth and division, it is important to know how protein syn- and then dissolved in 5 mM NaAc, pH 4.5, 0.5 M NaCl, and 10 mM thesis can be regulated. MgCl . Complete separation of tRNA from rRNA was achieved by gel Upon re-immersion of the quiescent dehydrated Artemia em- filtration on a Superose 12 FPLC column in the same buffer. The tRNA eluting at the position of tRNA from brewers’ yeast (Boehringer bryo in sea water, development quickly resumes. After a de- Mannheim) was pooled and reprecipitated with ethanol. The dried fined period of pre-emergence development, a free-swimming pellets were dissolved in 10 mM ammonium acetate, pH 4.5, and A nauplius with distinct morphological features emerges from its was measured. Samples containing 200 mg (3.7 A units) of tRNA shell. At the start of this period, protein synthesis and tran- were adjusted to pH 8 by addition of ammonium carbonate, pH 8, to scription resume quickly, both occurring in the absence of DNA 100 mM and incubated for1hat37 °Cto achieve the complete synthesis or cell division (1, 7). deacylation of aminoacyl-tRNA (19). Released amino acids were sep- arated from tRNA by ultrafiltration (Microcon-3, Amicon), modified by dimethylaminoazobenzene sulfonyl chloride treatment, separated by reverse phase chromatography on a C18 high performance liquid * This work was supported in part by a grant from the Netherlands chromatography column (100RP18e, Merck) and quantified at 436 nm Foundation of Chemical Research (SON). The costs of publication of this by comparison to known amounts of amino acids (20). When the tRNA article were defrayed in part by the payment of page charges. This samples were incubated at pH 4.5 instead of 8, no significant amount article must therefore be hereby marked “advertisement” in accordance of amino acid was found in the ultrafiltrate, indicating that our tRNA with 18 U.S.C. Section 1734 solely to indicate this fact. preparations are essentially devoid of contaminating free amino ‡ To whom correspondence should be addressed. Tel.: 31-71-5276131; Fax: 31-71-5276125. acids. 28912 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Charging of Transfer RNA during Development of Artemia 28913 Preparation of Extracts—Cell-free extracts from quiescent and devel- oping Artemia embryos were prepared precisely as described in Ref. 14. These extracts, referred to as “embryo lysate,” were used as such only for the in vitro translation experiments of Fig. 1. In the other experi- ments, the endogenous amino acid and nucleotide pools were removed from these extracts by passage of 2 ml of embryo lysate through a G-25 column (20 ml), previously equilibrated in 20 mM Hepes, pH 7.6, 100 mM potassium acetate, 1 mM magnesium acetate, 6 mM DTE, and 10% glycerol. Void-volume fractions, termed S30 extract, were pooled and stored in aliquots at 280 °C. The 100,000 3 g supernatant (S100) and 0.5 M KCl-washed (poly)ribosomal fraction as used in Fig. 4 were ob- tained from S30 extracts as described (17). Preparation of [ H]Val-labeled Aminoacyl-tRNA—Aminoacyl-tRNA from developing embryos was first deacylated as described above and separated from released amino acids by gel filtration on Superose 12. Next, 1 mg of the deacylated tRNA was recharged by an incubation for 12 min at 27 °C in 1 ml of 20 mM Tris-Cl, pH 7.4, 5 mM Mg Cl, 150 mM KCl, 0.5 mM DTE, 3 mM ATP, 10 mM phosphocreatine, 25 units of creatine phosphokinase, 0.1 mM each amino acid, and in the presence of FIG.1. Characterization of in vitro translation in Artemia. A, 400 mg of S100 protein from developing embryos and 200 mCi of [ H]Val. activation of protein synthesis during development. B, time-dependent The aminoacyl-tRNAs were re-extracted with phenol as described (17) amino acid incorporation in extracts from quiescent (E) and 20-h devel- and purified by gel filtration on Superose 12. Judged from the [ H]Val oping (l) embryos. C, mixing experiment: defined volumes of extract incorporation of 1.09 nmol of Val/20.8 nmol of tRNA, each tRNA species from 20-h developing embryos (0, 1, 2, 3, and 4 ml; 32 mg/ml) were was assumed to be fully charged with its cognate amino acid. adjusted to 4.0 ml with either buffer (l) or extract from quiescent In Vitro Translation—The rate of in vitro translation was measured embryos (E) (4, 3, 2, 1, and 0 ml, respectively; 31 mg/ml) and their in an assay (40 ml) containing 20 mM Hepes, pH 7.6, 50 mM potassium protein synthetic activity measured. Each point in A–C represents the incorporation of [ H]Val into protein per 7-ml aliquot during a 60-min acetate, 1.25 mM magnesium acetate, 0.1 mM spermidine, 0.2 mM DTE, (except in B) incubation at 27 °C. The reactions were performed with 0.2 mM both GTP and ATP, 500 units/ml RNasin (Promega), 10 mM embryo lysate (45 mg of protein) without added ATP, GTP, or unlabeled phosphocreatine, 25 units/ml creatine phosphokinase (Boehringer amino acids. Addition of up to 1 mM ATP did not affect incorporation, Mannheim), 3 mCi of L-[3,4- H]Val (specific radioactivity 10 Ci/mmol; indicating that the endogenous level of ATP together with the ATP- Amersham), unlabeled L-amino acids (35 mM each, except Val), and regenerating system is sufficient to sustain efficient translation in the extract as indicated in the legends for Figs. 1 and 4 (14). Reactions were 20-h developing embryo extract (14). The results of representative ex- performed at 27 °C, and at indicated times, 7-ml aliquots were with- periments are shown. drawn, immediately added to 150 ml of ice-cold 10% (w/v) trichloroacetic acid, and heated for 15 min at 95 °C. The precipitate was collected on a Miscellaneous Procedures—Protein concentration was measured by glass fiber filter (GF/C; Whatman) and washed with three 2-ml aliquots the method of Lowry et al. (25). of 10% trichloroacetic acid. The filter was dried at 95 °C (30 min) and its radioactivity measured in a liquid scintillation spectrometer. RESULTS Conditions for globin synthesis were the same, except for the follow- General Characteristics of in Vitro Translation during Devel- ing. Globin mRNA (2.5 mg; Life Technologies, Inc.) was added to micro- opment of Artemia Embryos—The rate of in vitro translation of coccal nuclease-treated extracts (see below), and the complete reaction endogenous mRNAs is low in quiescent embryo extracts, but mixture (40 ml) was processed to determine the incorporation of [ H]Val into protein. Incorporation was found to be time-dependent and globin increases strongly throughout pre-emergence development mRNA concentration-dependent with an optimum at 2.5 mg of globin (Fig. 1A). In Fig. 1B, the time courses of [ H]Val incorporation mRNA. into protein by extracts prepared from quiescent and 20-h Poly(Phe) Synthesis—Poly(U)-directed poly(Phe) synthesis was developing embryos are compared. The latter exhibits a fairly adapted from (21). To remove the endogenous mRNA pool completely, steep incorporation throughout the whole incubation period, S30 extracts (30 ml) were first treated for 15 min at 20 °C with 1 unit of which is in excellent agreement with the results of De Haro’s micrococcal nuclease (Pharmacia) in the presence of 1 mM CaCl . The nuclease was then inactivated by the addition of EGTA to a final group, where [ S]Met was used as a label (14). The incorpora- 3 3 concentration of 3 mM. Poly(Phe) synthesis was performed in a reaction tion of [ H]Phe and [ H]Leu into protein proceeds at a compa- mixture (40 ml) containing 20 mM Hepes, pH 7.6, 50 mM potassium rable rate. acetate, 1.25 mM magnesium acetate, 0.1 mM spermidine, 0.2 mM DTE, Concerning the nature of the process of reactivation of 0.2 mM both GTP and ATP, 500 units/ml RNasin (Promega), 10 mM protein synthesis in Artemia, translational repressors and phosphocreatine, 25 units/ml creatine phosphokinase (Boehringer), 5 activators have been postulated to be present in quiescent A units of poly(U)-programmed 80 S ribosomes from Artemia quies- cent embryos (21), and nuclease-treated extract (20 mg of protein). The and developing embryos, respectively (15). However, when labeled compound was either Artemia [ H]Phe-tRNA (40 pmol; 850 we mixed extracts prepared from quiescent and 20-h devel- cpm/pmol), previously labeled with L-[2,3,4,5,6- H]Phe using extract oping embryos in different ratios, we did not observe any from developing embryos as a source of phenylalanyl-tRNA synthetase effect of the extract from quiescent embryos on the transla- and reisolated as described (17), or 3 mCi of L-[2,3,4,5,6- H]Phe (specific tional activity of that of the developing embryos, nor the radioactivity 10 Ci/mmol, Amersham) together with deacylated tRNA reverse. In fact, the extract prepared from quiescent embryos from developing Artemia embryos (40 pmol). Reactions were performed at 27 °C, and 7-ml aliquots were withdrawn at specified times and behaves as a buffer (Fig. 1C). Thus, our results do not indi- processed as described above. cate the presence of a strong translational repressor in the Aminoacyl-tRNA Synthetase and Elongation Factor-1a Assays— quiescent nor an activator in the developing embryo, but Aminoacyl-tRNA synthetase activity was measured as described (22, rather suggest a shortage of one or several active transla- 23). The amount of ValRS in extracts was analyzed by Western blotting, tional components, which gradually get replenished during using an antiserum against Artemia ValRS (22, 24). The amount of development of the quiescent embryo. ValRS mRNA was determined by Northern blotting, using a ValRS- specific cDNA probe, prepared by polymerase chain reaction and stand- Changes in in Vivo tRNA Charging Levels during Develop- ard cloning techniques (18). Elongation factor-1a-dependent binding of ment—We used direct amino acid analysis on purified amino- [ H]Phe-tRNA to poly(U)-programmed 80 S ribosomes was performed acyl-tRNA to determine the degree of in vivo aminoacylation. as a control in the same extracts as described (17). The purification procedure includes homogenization of em- bryos in phenol at pH 4.5 to prevent deacylation, isolation of total RNA, followed by precipitation with lithium chloride to The abbreviations used are: DTE, dithioerythritol; ValRS, valyl- tRNA synthetase; poly(U), poly(uridylic acid). remove the major part of rRNA (26), and final purification of 28914 Charging of Transfer RNA during Development of Artemia FIG.2. Aminoacylation levels of transfer RNA during development. Charging is expressed as the amount of each individual amino acid attached to 2 nmol of total tRNA from Artemia embryos at different times of development (0, 1, and 20 h). Assuming that each of the 20 tRNA species represents 5% of the total tRNA population, 100 pmol of a specific amino acid/2 nmol of total tRNA equals a 100% charging level for this type of spe- cific tRNA. The result of a single repre- sentative experiment is shown. tRNA by gel filtration (see also Ref. 19). Compared with the nine) may be represented by two subsequent reactions method of periodate oxidation, the major advantage of this (Reaction 1). procedure is that the charging degree of all 20 different tRNA Phe-tRNA synthetase species can be assessed in a single experiment. 3 3 [ H]Phe 1 tRNA -|0 [ H]Phe-tRNA Neither the total yield of tRNA nor the ratio of tRNA to elongation factors rRNA changed significantly during development. The average | - 0 poly~@ H#Phe! 1 tRNA yield of 4.4 6 0.8 mg (n 5 6) tRNA/25 g of dried embryos is in the expected order when considering the EF-1a content of 22 REACTION 1 mg/25 g of embryos (27) and assuming equal molar amounts of EF-1a and tRNA present in Artemia (28). Moreover, the amino When the substrates [ H]Phe and uncharged tRNA are acid acceptance of tRNA prepared from quiescent and 20-h added separately, the rate of poly(Phe) synthesis is expected to developing embryos was the same for valine, leucine, lysine, depend on the amounts of elongation factors and phenylalanyl- phenylalanine, and glycine, when using nauplius S100 as a tRNA synthetase present in the extracts. In this case, the source of aminoacyl-tRNA synthetases. In the case of valine, activity of the quiescent embryo extract is found to be about both tRNA pools could be charged to a maximum of 2.7% of the 2.5-fold lower than that of the developing embryo extract (Fig. total tRNA pool by purified ValRS from Artemia (22). Under 3A). However, when charged [ H]Phe-tRNA is used instead of Val the assumption that tRNA represents 5% of the total tRNA [ H]Phe and tRNA, the difference in poly(Phe) synthesis be- pool, this indicates that at least half of the valine-accepting tween quiescent and developing embryo extracts disappears ends are intact, both in quiescent and developing embryos. We completely (Fig. 3B). The results of Fig. 3B indicate that elon- conclude that neither the total amount of tRNA nor its proc- gation factors 1 and 2 are equally active in quiescent and essing is significantly elevated during development. developing embryos. We conclude that a shortage of charged As seen from Fig. 2, there is a dramatic increase in the phenylalanyl-tRNA limits the elongation phase of protein syn- charging degree of each of the 20 tRNA-species during the thesis in extracts of quiescent embryos, at least under the first 20 h of development, from an average of 4 6 4% (n 5 3) direction of a synthetic messenger. in quiescent to 52 6 3% (n 5 2) in 20-h developing embryos. The Role of tRNA Aminoacylation in the Reactivation of The mutual relationship between the charging degree of Protein Synthesis—The extent to which total cell-free protein tRNA and the rate of translation is obvious, especially when synthesis of endogenous mRNA is limited by the shortage of taking into account that more than half of the 20 different aminoacyl-tRNAs was assessed as follows. In the presence of tRNA species are not charged at all in the quiescent embryo the two separate substrates, amino acids and total tRNA, the (detection limit 2% charging). However, not all tRNAs appear rate of protein synthesis in the quiescent embryo extract is Glu to be recharged in concert. After 1 h, tRNA already shows about 16 times lower as that observed in the developing em- Trp 40% charging, whereas tRNA after this time hardly car- bryo, i.e. the relative rate is 6.4 6 1.3% (mean 6 S.D., n 5 3) ries any amino acid (Fig. 2). Therefore, some aminoacyl-tRNA (Fig. 4A). Addition of the full complement of aminoacyl-tRNAs, species may clearly contribute more to the repression of over- however, markedly enhanced in vitro protein synthesis of the all protein synthesis in quiescent embryo lysates than others. quiescent embryo from a relative rate of 6.4 6 1.3% to a relative On the whole, the results of Figs. 1 and 2 clearly demonstrate rate of 23.6 6 1.2% (Fig. 4B). This proves that a paucity of a positive correlation between the capacity for protein syn- charged aminoacyl-tRNA significantly limits protein synthesis thesis and the degree of tRNA aminoacylation, and therefore in extracts of the quiescent embryo. support a model in which the arrest of translation in quies- Since protein synthesis could not be further stimulated by up cent Artemia embryos is based on a shortage of its natural to 20 mM aminoacyl-tRNAs, a second restriction in translation substrate, aminoacyl-tRNA. of available mRNAs appears to be present in the quiescent The Role of tRNA Aminoacylation in Poly(phenylalanine) embryo. Note that the level of mRNAs is comparable in both Synthesis—Whether the observed paucity of aminoacyl-tRNA types of embryo extracts (9). As a first attempt, we fractionated actually restricts elongation can be determined experimentally the quiescent and developing embryo extracts into the cytosolic by using the poly(U)-directed poly(phenylalanine) synthesis (S100), polyribosomal, and ribosomal wash fraction. Interest- 3 3 assay. The flow of [ H]phenylalanine into poly ([ H]phenylala- ingly, the purified polyribosomal fraction of developing em- Charging of Transfer RNA during Development of Artemia 28915 FIG.3. Aminoacyl-tRNAs restrict the elongation phase of pro- tein synthesis. A, poly(Phe) synthesis on added poly(U)-programmed ribosomes by extracts from quiescent (E) and 20-h developing (l) em- bryos using the separate substrates [ H]Phe and uncharged tRNA. B, the same as in A except that Artemia [ H]Phe-tRNA was used instead. Phe Comparable results were obtained with yeast tRNA instead of Ar- temia tRNA, although net incorporation was lower in this case (22). Incorporation is expressed in picomoles/mg of S30 protein. In the 20-h embryo extract, the rate of in vitro translation on endogenous mRNA was estimated to be 0.7 6 0.2 amino acid/s/ribosome, which is close to the in vivo rate of 2.5 amino acids/s/ribosome in rabbit reticulocytes (41). bryos was quite effective in stimulating protein synthesis of the quiescent embryo extract (S30), while the other fractions, in- cluding the ribosomes from quiescent embryos, were not. In fact, the polyribosomes from developing embryos activate pro- FIG.4. Reactivation of protein synthesis. A, in vitro protein syn- tein synthesis of the quiescent embryo S100 to a relative rate thesis on endogenous mRNAs by S30 extracts (30 mg of protein per 7 ml) 23.4 6 1.6% of that of the developing embryo S100 (Fig. 4C). of quiescent (E) and 20-h developing (l) embryos with the separate Ultimately, the further addition of fully charged aminoacyl- substrates, amino acids (35 mM each, except 10 mM [ H]Val) and un- charged tRNA (2.5 mg). B, the same as in A except that fully charged tRNAs completely restored protein synthesis in the extract of aminoacyl-tRNA (2.5 mg; labeled with [ H]Val) was used instead of the the quiescent embryo to the level of the developed embryo (Fig. separate substrates. C, the same as in A except that S100 (17.5 mgof 4D). Moreover, the ribosomal wash fractions, which contained protein) was used instead of S30, together with the purified ribosomes large amounts of initiation factors (10), were without effect, (15 mg of protein) from developing embryos. D, the same as in C except thus indicating that they were not rate-limiting. As shown that fully charged aminoacyl-tRNA (2.5 mg; labeled with [ H]Val) was used instead of the separate substrates. Since during S100 preparation previously by others, initiation factor eIF-2 was found to re- the protein concentration decreased from ;30 mg/ml in S30 to ;17.5 main constant in amount and activity during development (10). mg/ml in S100, the amount of S100 protein was reduced proportionally. A substantial effect of charged tRNAs was also seen on the Assays were optimized with respect to the amount of ribosomes re- translation of globin mRNA by the quiescent embryo extract. quired. Initial rates were calculated from the data of the first 15 min. Addition of the full complement of aminoacyl-tRNAs to RNase- treated extracts of the quiescent embryo raises the synthesis of globin from a relative rate of 34 6 8% to a relative rate of 76 6 aminoacyl-tRNA synthetase 10%. Absolute values, however, were an order of magnitude aa 1 tRNA 1 ATP -|0 aa-tRNA 1 AMP 1 PP less than those observed with polyribosomes in the two types of extract. This was also the case with translation of poly(A) REACTION 2 mRNAs extracted from the developing embryo, indicating that The tRNA pool of the quiescent embryo itself appears to be “naked” mRNA is a poor substrate for translation by the qui- comparable to that of the developing embryo (this study and escent and the developing embryo extract as well. We conclude, Ref. 16). The level of endogenous amino acids rises only about therefore, that during the development of Artemia embryos, 2-fold (Ref. 1 and our own observations), while the ATP con- protein synthesis is activated by the recharging of aminoacyl- centration is cited to increase 7-fold during development (29). tRNAs, but due to the virtual absence of polyribosomes in the However, the shortage of ATP by itself is not responsible for the quiescent embryo (1, 5, 13), the rate of protein synthesis re- inhibition of translation, since we found that the addition of mains lower than in the developing embryo. ATP up to 1 mM does not restore protein synthesis in quiescent Levels of Aminoacyl-tRNA Synthetases during Develop- embryo extracts. Therefore, we investigated instead the behav- ment—Upon rehydration of the quiescent embryos, tRNA may ior of three selected aminoacyl-tRNA synthetases, all of which be recharged by its cognate aminoacyl-tRNA synthetase as were found to rise during the first 20 h of development, i.e. present at this stage. However, the low degree of charging of most of the tRNAs even after1hof development (Fig. 2) valyl- and lysyl-tRNA synthetase by a factor of about 2, while indicates a severe limitation in the supply of aminoacyl-tRNAs. methionyl-tRNA synthetase increased more than 10-fold (Ta- Apparently, the aminoacylation reaction is limited by one or ble I). Elongation factor-1a activity, which was measured as a more of its reacting components. control, remained constant. 28916 Charging of Transfer RNA during Development of Artemia TABLE I developing embryos is largely due to the added (poly)ribosomal Aminoacyl-tRNA synthetase activities in extracts mRNAs rather than the endogenous mRNA. Together, these of quiescent and developing embryos observations confirm that the mRNA from developing embryos Aminoacyl-tRNA Specific activity is already loaded with ribosomes and translated as such by the synthetase Increase specific for Quiescent Developing quiescent embryo extract. Once the mRNA is initiated, the ribosomes elongate with a rate comparable to that in the de- units/mg units/mg -fold veloping embryo provided the restriction of charging of elon- Valine 43 67 1.6 gating tRNAs is released. Methionine 3.7 51 14 Lysine 180 400 2.2 It is known that phosphorylation is important in the regula- tion of the initiation step of protein synthesis (30), but so far Specific activities were calculated from the linear region of concen- tration-dependent plots obtained with extracts of quiescent and 20-h the evidence for phosphorylation playing an important role in developing embryo, incubated for 10 min at 25 °C in the presence of 3.5 synthetase function has been less conclusive, at least for ver- mg/ml yeast tRNA and H-labeled amino acid. One unit of activity tebrates (32). We prefer to think that a deficiency in the abso- corresponds to the formation of 1 pmol of aminoacyl-tRNA/min. lute amount of these enzymes may be responsible for the low synthetase activity in quiescent embryo extracts, as is also the DISCUSSION case in Bombyx mori (33). In fact, at least for ValRS, we have It is generally agreed that the rate-limiting steps in protein found by Western blotting that the amount of the protein synthesis lies at the level of initiation (30). The same situation increases significantly during development with retention of its does not necessarily prevail to reactivate protein synthesis intrinsic specificity. Since Northern blot analysis also reveal after a period of quiescence like in the brine shrimp Artemia. that the level of ValRS mRNA markedly increases during de- For instance, efforts to prove deficiencies in the initiation fac- velopment, the de novo synthesis of the ValRS enzyme may be tors of quiescent embryos have been rather unconvincing (31), attributed to enhanced transcription of the ValRS gene. It and levels of the initiation factor eIF-2 are found to be the same should be stressed that although the synthetase levels in qui- before and after development (10). Most characteristic of qui- escent embryos so far are substantial, apparently they are not escent embryos, however, is the inactive form of its mRNAs and high enough to recharge the pool of deacylated tRNAs within a the slow reappearance of polysomes during development (9, period of minutes, as in rapidly growing Escherichia coli and 13). However, this by itself does not explain that the encapsu- the re-activated spores of Bacillus megaterium (34). Synthetase lated embryo exhibits no protein synthesis unless resumption levels can never become zero in the quiescent embryo; they of development ensues. In this context, we have proven the should be low enough to prevent full charging, but high enough absence of an inhibitor, although adequate levels of mRNAs are to enable their own synthesis, because otherwise protein syn- present in such extracts (9). How protein synthesis is reacti- thesis would be irreversibly arrested. Careful studies on the vated during development of Artemia has therefore remained a transcriptional regulation of aminoacyl-tRNA synthetases dur- long-standing question (8 –15). We demonstrate that during ing development are therefore indicated. the development of quiescent embryos into metabolically active The aminoacylation of tRNA appears to function as a sensor embryos there is a dramatic increase in the level of tRNA that regulates the biosynthesis of amino acids via transcription aminoacylation. We also establish that the difference in protein factor GCN4 in yeast (35) and also the transcription of amino- synthesis capacity between extracts from quiescent and devel- acyl-tRNA synthetase genes in Gram-positive bacteria (36, 37). oping embryos can be completely abolished by solely adding We have demonstrated here that protein synthesis itself is fully charged aminoacyl-tRNA, together with the mixture of controlled at the level of tRNA charging during the natural polyribosomes of developing embryos as an external source of development of a eukaryote. Interestingly, in bacterial spores, translatable mRNA. Furthermore, we show by poly(U)-directed the level of tRNA charging is also very low and increases poly(Phe) synthesis that only the shortage of charged amino- rapidly upon germination (34). Moreover, tRNAs specific for acyl-tRNA restricts the elongation step of the quiescent em- valine, arginine, and histidine are among the most significantly bryo. In our opinion, our data prove for the first time that a charged tRNAs in these spores, as also seems to be the case in shortage of charged aminoacyl-tRNA in combination with a low Artemia embryos. Furthermore, studies on E. coli and mouse level of preformed polyribosomes is the cause of the extreme ascites tumor cells show that protein synthesis decreases when low rate of protein synthesis in extracts of the quiescent the charging degree of tRNAs is lowered on purpose (38, 39). embryo. Aminoacylation of tRNAs therefore appears to play a general The question which immediately comes to mind is what regulatory role in escaping routes from dormancy and may also regulates the charging of tRNAs in vivo during Artemia devel- be important for instance in germination of plant seeds and opment? Rather than addressing here how the charging level of perhaps also in the revival of G -arrested vertebrate cells. tRNAs have become low in the quiescent embryo, we focus on Since the present day adaptor molecule, tRNA, is believed to their reactivation after release from quiescence. Because we have been of prime importance in the genesis of protein syn- have shown that at least some of the aminoacyl-tRNA syn- thesis and life itself (40), the regulation of protein synthesis thetases, especially methionyl-tRNA synthetase, display an el- may even have started at the level of tRNA aminoacylation, evated activity in developing embryo lysates, they are prime while fine tuning of mRNA expression at the level of ribosome- candidates for the control of protein synthesis in Artemia. This induced initiation and elongation events occurred later on in immediately places the initiator tRNA in the limelight, and the evolution. Met shortage of charged initiator tRNA could explain the low Acknowledgments—We thank Dr. R. Amons and Dr. J. Dijk for their fraction of ribosomes, present as polysomes in quiescent em- expert advice and Dr. B. Kraal and Dr. C. W. A. Pleij for critically bryos (5, 9, 13). In fact, we have found that the polyribosomal reading the manuscript. fraction from developing embryos contains about 3 times more poly(A) RNA than that of quiescent embryos, in agreement REFERENCES with the observed shift from monosomes to polysomes at the 1. Clegg, J. S., and Conte, F. P. (1980) in The Brine Shrimp Artemia (Persoone, G., Sorgeloos, P., Roels, O., and Jaspers, E., eds) Vol. 2, pp. 11–54, Universa onset of development (5, 13). Press, Wetteren, Belgium Moreover, we found that the observed activation of the qui- 2. Lavens, P.and Sorgeloos, P. (1987) in Artemia Research and Its Applications escent embryo extract by the (poly)ribosomal fraction from (Decleir, W., Moens, L., Slegers, H., Sorgeloos, P., and Jaspers, E., eds) Vol. Charging of Transfer RNA during Development of Artemia 28917 3, pp. 27– 63, Universa Press, Wetteren, Belgium Analysis (Wittmann-Liebold, B., Salnikow, J., and Erdmann, V. A., eds) pp. 3. Crowe, J. H., Crowe, L. M., Drinkwater, L., and Busa, W. B. (1987) in Artemia 56 – 61, Springer-Verlag, Berlin Research and Its Applications (Decleir, W., Moens, L., Slegers, H., 21. Iwasaki, K., and Kaziro, Y. (1981) in RNA and Protein Synthesis (Moldave, K., Sorgeloos, P.and Jaspers, E., eds) Vol. 2, pp. 19 – 40, Universa Press, ed) pp. 435– 454, Academic Press, New York Wetteren, Belgium 22. Brandsma, M., Kerjan, P., Dijk, J., Janssen, G. M. C., and Mo ¨ ller, W. (1995) 4. Clegg, J. S., Drinkwater, L. E., and Sorgeloos, P. (1996) Physiol. Zool. 69, Eur. J. Biochem. 233, 277–282 49–66 23. 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(1979) Biochim. Biophys. Acta 565, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold 293–304 Spring Harbor, NY 19. Tockman, J., and Vold, B. S. (1977) J. Bacteriol. 130, 1091–1097 40. Crick, F. H. C. (1968) J. Mol. Biol. 38, 367–379 20. Knecht, R.and Chang, J. (1986) in Advanced Methods in Protein Microsequence 41. Dintzis, H. M. (1961) Proc. Natl. Acad. Sci. U. S. A. 47, 247–261 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Termination of Quiescence in Crustacea

Brandsma, Margreet; Janssen, George M.C.; Möller, Wim
Journal of Biological Chemistry – Nov 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 46, Issue of November 14, pp. 28912–28917, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. THE ROLE OF TRANSFER RNA AMINOACYLATION IN THE BRINE SHRIMP ARTEMIA* (Received for publication, July 31, 1997) Margreet Brandsma, George M. C. Janssen, and Wim Mo ¨ ller‡ From the Department of Medical Biochemistry, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands After 30 years of research on the re-activation of translation In quiescent embryos of the brine shrimp Artemia, the level of aminoacylation of transfer RNAs is low. in Artemia, it is clear that quiescent and developing embryos During resumption of development the charging level contain approximately equal amounts of ribosomes (8), mRNA of transfer RNAs increases, concomitant with the acti- (9), initiation factors (10), elongation factors (11), and termina- vation of protein synthesis. The total level of charging tion factors (12), which are all equally active in in vitro trans- rises dramatically from an average of 4% to 50% within lation assays. At the onset of development, a slow but definite a period of 24 h of development. The restriction of in shift from 80 S ribosomes to polysomes can be observed (5, 13). vitro translation of the quiescent embryo extract can However, extracts from quiescent embryos are inactive in be partially released by the addition of charged ami- translation of their endogenous mRNAs (14). This paradox has noacyl-tRNA, which apparently starts the flow of ribo- led several investigators to search for specific inhibitors and somes into polyribosome structures. Complete reacti- activators of translation in extracts from non-developing and vation of translation by aminoacyl-tRNA occurs when developing embryos, respectively (15). Until now, none of these mRNA from preformed mRNA-ribosome complexes, approaches has provided a definite answer. like the polyribosomes extracted from developing em- Since ribosomes, factors, and mRNAs appear to be normal in bryos or poly(U)-programmed ribosomes, are offered the quiescent embryo, we have explored the level of charged to quiescent embryo extracts. With respect to the tRNA during development. Although the amount of tRNA as mechanism of in vivo recharging of tRNAs, we ob- such does not change significantly during development (16), served that the level of several aminoacyl-tRNA syn- the degree of tRNA aminoacylation has until now received little thetases increase during development. Methionyl- attention. The results of such studies are presented here. tRNA synthetase rises more than 10-fold. In the case of valyl-tRNA synthetase, the activation is lower and EXPERIMENTAL PROCEDURES shown to be due to the de novo synthesis of its mRNA Culturing Conditions—Dried Artemia embryos (San Francisco Bay and the corresponding protein product as well. We Brand Inc., San Francisco, CA) were washed with 2% NaOCl as de- conclude that protein synthesis and thereby the grad- scribed (17) and either used directly (quiescent embryos) or cultured for ual animation of cryptobiotic Artemia embryos is de- the time indicated in aerated artificial sea water at 27 °C (developing termined to a large extent by the rate by which ami- embryos). Under these conditions, approximately 80% of the embryos noacyl-tRNAs are replenished during development at emerge from their shells within 20 h. both the initiation and elongation level. In Vivo tRNA Charging Levels—Dried embryos (25 g) were cul- tured for the time indicated and washed with distilled water and 50 mM NaAc, pH 4.5, 150 mM NaCl. The volume was adjusted to 75 ml with the same buffer, 50 ml of phenol (saturated with the same Multicellular organisms like the crustacean Artemia can sur- buffer) was added, and this mixture was homogenized three times for vive long periods of environmental stress by entering a crypto- 45 s with a Polytron homogenizer. Upon centrifugation for 12 min at biotic stage (see Refs. 1– 4 for reviews). Tissue culture cells can 3000 3 g in a Beckman JA 10 rotor, the aqueous (upper) phase was protect themselves against adverse growing conditions by en- extracted two more times with the same amount of acidic phenol. tering the G phase of the cell cycle. In both cases, the rate of RNA from the final upper phase was precipitated with ethanol (18). The dried pellet was dissolved in 15 ml of 10 mM NaAc, pH 4.5, and protein synthesis is reduced to a low level and its reactivation lithium chloride was added to a concentration of 0.8 M, followed by is a prerequisite for the cells to re-enter the cell cycle (5, 6). For centrifugation for 20 min at 9000 3 g in a Sorvall SS 34 rotor. The a better understanding of the processes that determine cell tRNA present in the supernatant was reprecipitated with ethanol growth and division, it is important to know how protein syn- and then dissolved in 5 mM NaAc, pH 4.5, 0.5 M NaCl, and 10 mM thesis can be regulated. MgCl . Complete separation of tRNA from rRNA was achieved by gel Upon re-immersion of the quiescent dehydrated Artemia em- filtration on a Superose 12 FPLC column in the same buffer. The tRNA eluting at the position of tRNA from brewers’ yeast (Boehringer bryo in sea water, development quickly resumes. After a de- Mannheim) was pooled and reprecipitated with ethanol. The dried fined period of pre-emergence development, a free-swimming pellets were dissolved in 10 mM ammonium acetate, pH 4.5, and A nauplius with distinct morphological features emerges from its was measured. Samples containing 200 mg (3.7 A units) of tRNA shell. At the start of this period, protein synthesis and tran- were adjusted to pH 8 by addition of ammonium carbonate, pH 8, to scription resume quickly, both occurring in the absence of DNA 100 mM and incubated for1hat37 °Cto achieve the complete synthesis or cell division (1, 7). deacylation of aminoacyl-tRNA (19). Released amino acids were sep- arated from tRNA by ultrafiltration (Microcon-3, Amicon), modified by dimethylaminoazobenzene sulfonyl chloride treatment, separated by reverse phase chromatography on a C18 high performance liquid * This work was supported in part by a grant from the Netherlands chromatography column (100RP18e, Merck) and quantified at 436 nm Foundation of Chemical Research (SON). The costs of publication of this by comparison to known amounts of amino acids (20). When the tRNA article were defrayed in part by the payment of page charges. This samples were incubated at pH 4.5 instead of 8, no significant amount article must therefore be hereby marked “advertisement” in accordance of amino acid was found in the ultrafiltrate, indicating that our tRNA with 18 U.S.C. Section 1734 solely to indicate this fact. preparations are essentially devoid of contaminating free amino ‡ To whom correspondence should be addressed. Tel.: 31-71-5276131; Fax: 31-71-5276125. acids. 28912 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Charging of Transfer RNA during Development of Artemia 28913 Preparation of Extracts—Cell-free extracts from quiescent and devel- oping Artemia embryos were prepared precisely as described in Ref. 14. These extracts, referred to as “embryo lysate,” were used as such only for the in vitro translation experiments of Fig. 1. In the other experi- ments, the endogenous amino acid and nucleotide pools were removed from these extracts by passage of 2 ml of embryo lysate through a G-25 column (20 ml), previously equilibrated in 20 mM Hepes, pH 7.6, 100 mM potassium acetate, 1 mM magnesium acetate, 6 mM DTE, and 10% glycerol. Void-volume fractions, termed S30 extract, were pooled and stored in aliquots at 280 °C. The 100,000 3 g supernatant (S100) and 0.5 M KCl-washed (poly)ribosomal fraction as used in Fig. 4 were ob- tained from S30 extracts as described (17). Preparation of [ H]Val-labeled Aminoacyl-tRNA—Aminoacyl-tRNA from developing embryos was first deacylated as described above and separated from released amino acids by gel filtration on Superose 12. Next, 1 mg of the deacylated tRNA was recharged by an incubation for 12 min at 27 °C in 1 ml of 20 mM Tris-Cl, pH 7.4, 5 mM Mg Cl, 150 mM KCl, 0.5 mM DTE, 3 mM ATP, 10 mM phosphocreatine, 25 units of creatine phosphokinase, 0.1 mM each amino acid, and in the presence of FIG.1. Characterization of in vitro translation in Artemia. A, 400 mg of S100 protein from developing embryos and 200 mCi of [ H]Val. activation of protein synthesis during development. B, time-dependent The aminoacyl-tRNAs were re-extracted with phenol as described (17) amino acid incorporation in extracts from quiescent (E) and 20-h devel- and purified by gel filtration on Superose 12. Judged from the [ H]Val oping (l) embryos. C, mixing experiment: defined volumes of extract incorporation of 1.09 nmol of Val/20.8 nmol of tRNA, each tRNA species from 20-h developing embryos (0, 1, 2, 3, and 4 ml; 32 mg/ml) were was assumed to be fully charged with its cognate amino acid. adjusted to 4.0 ml with either buffer (l) or extract from quiescent In Vitro Translation—The rate of in vitro translation was measured embryos (E) (4, 3, 2, 1, and 0 ml, respectively; 31 mg/ml) and their in an assay (40 ml) containing 20 mM Hepes, pH 7.6, 50 mM potassium protein synthetic activity measured. Each point in A–C represents the incorporation of [ H]Val into protein per 7-ml aliquot during a 60-min acetate, 1.25 mM magnesium acetate, 0.1 mM spermidine, 0.2 mM DTE, (except in B) incubation at 27 °C. The reactions were performed with 0.2 mM both GTP and ATP, 500 units/ml RNasin (Promega), 10 mM embryo lysate (45 mg of protein) without added ATP, GTP, or unlabeled phosphocreatine, 25 units/ml creatine phosphokinase (Boehringer amino acids. Addition of up to 1 mM ATP did not affect incorporation, Mannheim), 3 mCi of L-[3,4- H]Val (specific radioactivity 10 Ci/mmol; indicating that the endogenous level of ATP together with the ATP- Amersham), unlabeled L-amino acids (35 mM each, except Val), and regenerating system is sufficient to sustain efficient translation in the extract as indicated in the legends for Figs. 1 and 4 (14). Reactions were 20-h developing embryo extract (14). The results of representative ex- performed at 27 °C, and at indicated times, 7-ml aliquots were with- periments are shown. drawn, immediately added to 150 ml of ice-cold 10% (w/v) trichloroacetic acid, and heated for 15 min at 95 °C. The precipitate was collected on a Miscellaneous Procedures—Protein concentration was measured by glass fiber filter (GF/C; Whatman) and washed with three 2-ml aliquots the method of Lowry et al. (25). of 10% trichloroacetic acid. The filter was dried at 95 °C (30 min) and its radioactivity measured in a liquid scintillation spectrometer. RESULTS Conditions for globin synthesis were the same, except for the follow- General Characteristics of in Vitro Translation during Devel- ing. Globin mRNA (2.5 mg; Life Technologies, Inc.) was added to micro- opment of Artemia Embryos—The rate of in vitro translation of coccal nuclease-treated extracts (see below), and the complete reaction endogenous mRNAs is low in quiescent embryo extracts, but mixture (40 ml) was processed to determine the incorporation of [ H]Val into protein. Incorporation was found to be time-dependent and globin increases strongly throughout pre-emergence development mRNA concentration-dependent with an optimum at 2.5 mg of globin (Fig. 1A). In Fig. 1B, the time courses of [ H]Val incorporation mRNA. into protein by extracts prepared from quiescent and 20-h Poly(Phe) Synthesis—Poly(U)-directed poly(Phe) synthesis was developing embryos are compared. The latter exhibits a fairly adapted from (21). To remove the endogenous mRNA pool completely, steep incorporation throughout the whole incubation period, S30 extracts (30 ml) were first treated for 15 min at 20 °C with 1 unit of which is in excellent agreement with the results of De Haro’s micrococcal nuclease (Pharmacia) in the presence of 1 mM CaCl . The nuclease was then inactivated by the addition of EGTA to a final group, where [ S]Met was used as a label (14). The incorpora- 3 3 concentration of 3 mM. Poly(Phe) synthesis was performed in a reaction tion of [ H]Phe and [ H]Leu into protein proceeds at a compa- mixture (40 ml) containing 20 mM Hepes, pH 7.6, 50 mM potassium rable rate. acetate, 1.25 mM magnesium acetate, 0.1 mM spermidine, 0.2 mM DTE, Concerning the nature of the process of reactivation of 0.2 mM both GTP and ATP, 500 units/ml RNasin (Promega), 10 mM protein synthesis in Artemia, translational repressors and phosphocreatine, 25 units/ml creatine phosphokinase (Boehringer), 5 activators have been postulated to be present in quiescent A units of poly(U)-programmed 80 S ribosomes from Artemia quies- cent embryos (21), and nuclease-treated extract (20 mg of protein). The and developing embryos, respectively (15). However, when labeled compound was either Artemia [ H]Phe-tRNA (40 pmol; 850 we mixed extracts prepared from quiescent and 20-h devel- cpm/pmol), previously labeled with L-[2,3,4,5,6- H]Phe using extract oping embryos in different ratios, we did not observe any from developing embryos as a source of phenylalanyl-tRNA synthetase effect of the extract from quiescent embryos on the transla- and reisolated as described (17), or 3 mCi of L-[2,3,4,5,6- H]Phe (specific tional activity of that of the developing embryos, nor the radioactivity 10 Ci/mmol, Amersham) together with deacylated tRNA reverse. In fact, the extract prepared from quiescent embryos from developing Artemia embryos (40 pmol). Reactions were performed at 27 °C, and 7-ml aliquots were withdrawn at specified times and behaves as a buffer (Fig. 1C). Thus, our results do not indi- processed as described above. cate the presence of a strong translational repressor in the Aminoacyl-tRNA Synthetase and Elongation Factor-1a Assays— quiescent nor an activator in the developing embryo, but Aminoacyl-tRNA synthetase activity was measured as described (22, rather suggest a shortage of one or several active transla- 23). The amount of ValRS in extracts was analyzed by Western blotting, tional components, which gradually get replenished during using an antiserum against Artemia ValRS (22, 24). The amount of development of the quiescent embryo. ValRS mRNA was determined by Northern blotting, using a ValRS- specific cDNA probe, prepared by polymerase chain reaction and stand- Changes in in Vivo tRNA Charging Levels during Develop- ard cloning techniques (18). Elongation factor-1a-dependent binding of ment—We used direct amino acid analysis on purified amino- [ H]Phe-tRNA to poly(U)-programmed 80 S ribosomes was performed acyl-tRNA to determine the degree of in vivo aminoacylation. as a control in the same extracts as described (17). The purification procedure includes homogenization of em- bryos in phenol at pH 4.5 to prevent deacylation, isolation of total RNA, followed by precipitation with lithium chloride to The abbreviations used are: DTE, dithioerythritol; ValRS, valyl- tRNA synthetase; poly(U), poly(uridylic acid). remove the major part of rRNA (26), and final purification of 28914 Charging of Transfer RNA during Development of Artemia FIG.2. Aminoacylation levels of transfer RNA during development. Charging is expressed as the amount of each individual amino acid attached to 2 nmol of total tRNA from Artemia embryos at different times of development (0, 1, and 20 h). Assuming that each of the 20 tRNA species represents 5% of the total tRNA population, 100 pmol of a specific amino acid/2 nmol of total tRNA equals a 100% charging level for this type of spe- cific tRNA. The result of a single repre- sentative experiment is shown. tRNA by gel filtration (see also Ref. 19). Compared with the nine) may be represented by two subsequent reactions method of periodate oxidation, the major advantage of this (Reaction 1). procedure is that the charging degree of all 20 different tRNA Phe-tRNA synthetase species can be assessed in a single experiment. 3 3 [ H]Phe 1 tRNA -|0 [ H]Phe-tRNA Neither the total yield of tRNA nor the ratio of tRNA to elongation factors rRNA changed significantly during development. The average | - 0 poly~@ H#Phe! 1 tRNA yield of 4.4 6 0.8 mg (n 5 6) tRNA/25 g of dried embryos is in the expected order when considering the EF-1a content of 22 REACTION 1 mg/25 g of embryos (27) and assuming equal molar amounts of EF-1a and tRNA present in Artemia (28). Moreover, the amino When the substrates [ H]Phe and uncharged tRNA are acid acceptance of tRNA prepared from quiescent and 20-h added separately, the rate of poly(Phe) synthesis is expected to developing embryos was the same for valine, leucine, lysine, depend on the amounts of elongation factors and phenylalanyl- phenylalanine, and glycine, when using nauplius S100 as a tRNA synthetase present in the extracts. In this case, the source of aminoacyl-tRNA synthetases. In the case of valine, activity of the quiescent embryo extract is found to be about both tRNA pools could be charged to a maximum of 2.7% of the 2.5-fold lower than that of the developing embryo extract (Fig. total tRNA pool by purified ValRS from Artemia (22). Under 3A). However, when charged [ H]Phe-tRNA is used instead of Val the assumption that tRNA represents 5% of the total tRNA [ H]Phe and tRNA, the difference in poly(Phe) synthesis be- pool, this indicates that at least half of the valine-accepting tween quiescent and developing embryo extracts disappears ends are intact, both in quiescent and developing embryos. We completely (Fig. 3B). The results of Fig. 3B indicate that elon- conclude that neither the total amount of tRNA nor its proc- gation factors 1 and 2 are equally active in quiescent and essing is significantly elevated during development. developing embryos. We conclude that a shortage of charged As seen from Fig. 2, there is a dramatic increase in the phenylalanyl-tRNA limits the elongation phase of protein syn- charging degree of each of the 20 tRNA-species during the thesis in extracts of quiescent embryos, at least under the first 20 h of development, from an average of 4 6 4% (n 5 3) direction of a synthetic messenger. in quiescent to 52 6 3% (n 5 2) in 20-h developing embryos. The Role of tRNA Aminoacylation in the Reactivation of The mutual relationship between the charging degree of Protein Synthesis—The extent to which total cell-free protein tRNA and the rate of translation is obvious, especially when synthesis of endogenous mRNA is limited by the shortage of taking into account that more than half of the 20 different aminoacyl-tRNAs was assessed as follows. In the presence of tRNA species are not charged at all in the quiescent embryo the two separate substrates, amino acids and total tRNA, the (detection limit 2% charging). However, not all tRNAs appear rate of protein synthesis in the quiescent embryo extract is Glu to be recharged in concert. After 1 h, tRNA already shows about 16 times lower as that observed in the developing em- Trp 40% charging, whereas tRNA after this time hardly car- bryo, i.e. the relative rate is 6.4 6 1.3% (mean 6 S.D., n 5 3) ries any amino acid (Fig. 2). Therefore, some aminoacyl-tRNA (Fig. 4A). Addition of the full complement of aminoacyl-tRNAs, species may clearly contribute more to the repression of over- however, markedly enhanced in vitro protein synthesis of the all protein synthesis in quiescent embryo lysates than others. quiescent embryo from a relative rate of 6.4 6 1.3% to a relative On the whole, the results of Figs. 1 and 2 clearly demonstrate rate of 23.6 6 1.2% (Fig. 4B). This proves that a paucity of a positive correlation between the capacity for protein syn- charged aminoacyl-tRNA significantly limits protein synthesis thesis and the degree of tRNA aminoacylation, and therefore in extracts of the quiescent embryo. support a model in which the arrest of translation in quies- Since protein synthesis could not be further stimulated by up cent Artemia embryos is based on a shortage of its natural to 20 mM aminoacyl-tRNAs, a second restriction in translation substrate, aminoacyl-tRNA. of available mRNAs appears to be present in the quiescent The Role of tRNA Aminoacylation in Poly(phenylalanine) embryo. Note that the level of mRNAs is comparable in both Synthesis—Whether the observed paucity of aminoacyl-tRNA types of embryo extracts (9). As a first attempt, we fractionated actually restricts elongation can be determined experimentally the quiescent and developing embryo extracts into the cytosolic by using the poly(U)-directed poly(phenylalanine) synthesis (S100), polyribosomal, and ribosomal wash fraction. Interest- 3 3 assay. The flow of [ H]phenylalanine into poly ([ H]phenylala- ingly, the purified polyribosomal fraction of developing em- Charging of Transfer RNA during Development of Artemia 28915 FIG.3. Aminoacyl-tRNAs restrict the elongation phase of pro- tein synthesis. A, poly(Phe) synthesis on added poly(U)-programmed ribosomes by extracts from quiescent (E) and 20-h developing (l) em- bryos using the separate substrates [ H]Phe and uncharged tRNA. B, the same as in A except that Artemia [ H]Phe-tRNA was used instead. Phe Comparable results were obtained with yeast tRNA instead of Ar- temia tRNA, although net incorporation was lower in this case (22). Incorporation is expressed in picomoles/mg of S30 protein. In the 20-h embryo extract, the rate of in vitro translation on endogenous mRNA was estimated to be 0.7 6 0.2 amino acid/s/ribosome, which is close to the in vivo rate of 2.5 amino acids/s/ribosome in rabbit reticulocytes (41). bryos was quite effective in stimulating protein synthesis of the quiescent embryo extract (S30), while the other fractions, in- cluding the ribosomes from quiescent embryos, were not. In fact, the polyribosomes from developing embryos activate pro- FIG.4. Reactivation of protein synthesis. A, in vitro protein syn- tein synthesis of the quiescent embryo S100 to a relative rate thesis on endogenous mRNAs by S30 extracts (30 mg of protein per 7 ml) 23.4 6 1.6% of that of the developing embryo S100 (Fig. 4C). of quiescent (E) and 20-h developing (l) embryos with the separate Ultimately, the further addition of fully charged aminoacyl- substrates, amino acids (35 mM each, except 10 mM [ H]Val) and un- charged tRNA (2.5 mg). B, the same as in A except that fully charged tRNAs completely restored protein synthesis in the extract of aminoacyl-tRNA (2.5 mg; labeled with [ H]Val) was used instead of the the quiescent embryo to the level of the developed embryo (Fig. separate substrates. C, the same as in A except that S100 (17.5 mgof 4D). Moreover, the ribosomal wash fractions, which contained protein) was used instead of S30, together with the purified ribosomes large amounts of initiation factors (10), were without effect, (15 mg of protein) from developing embryos. D, the same as in C except thus indicating that they were not rate-limiting. As shown that fully charged aminoacyl-tRNA (2.5 mg; labeled with [ H]Val) was used instead of the separate substrates. Since during S100 preparation previously by others, initiation factor eIF-2 was found to re- the protein concentration decreased from ;30 mg/ml in S30 to ;17.5 main constant in amount and activity during development (10). mg/ml in S100, the amount of S100 protein was reduced proportionally. A substantial effect of charged tRNAs was also seen on the Assays were optimized with respect to the amount of ribosomes re- translation of globin mRNA by the quiescent embryo extract. quired. Initial rates were calculated from the data of the first 15 min. Addition of the full complement of aminoacyl-tRNAs to RNase- treated extracts of the quiescent embryo raises the synthesis of globin from a relative rate of 34 6 8% to a relative rate of 76 6 aminoacyl-tRNA synthetase 10%. Absolute values, however, were an order of magnitude aa 1 tRNA 1 ATP -|0 aa-tRNA 1 AMP 1 PP less than those observed with polyribosomes in the two types of extract. This was also the case with translation of poly(A) REACTION 2 mRNAs extracted from the developing embryo, indicating that The tRNA pool of the quiescent embryo itself appears to be “naked” mRNA is a poor substrate for translation by the qui- comparable to that of the developing embryo (this study and escent and the developing embryo extract as well. We conclude, Ref. 16). The level of endogenous amino acids rises only about therefore, that during the development of Artemia embryos, 2-fold (Ref. 1 and our own observations), while the ATP con- protein synthesis is activated by the recharging of aminoacyl- centration is cited to increase 7-fold during development (29). tRNAs, but due to the virtual absence of polyribosomes in the However, the shortage of ATP by itself is not responsible for the quiescent embryo (1, 5, 13), the rate of protein synthesis re- inhibition of translation, since we found that the addition of mains lower than in the developing embryo. ATP up to 1 mM does not restore protein synthesis in quiescent Levels of Aminoacyl-tRNA Synthetases during Develop- embryo extracts. Therefore, we investigated instead the behav- ment—Upon rehydration of the quiescent embryos, tRNA may ior of three selected aminoacyl-tRNA synthetases, all of which be recharged by its cognate aminoacyl-tRNA synthetase as were found to rise during the first 20 h of development, i.e. present at this stage. However, the low degree of charging of most of the tRNAs even after1hof development (Fig. 2) valyl- and lysyl-tRNA synthetase by a factor of about 2, while indicates a severe limitation in the supply of aminoacyl-tRNAs. methionyl-tRNA synthetase increased more than 10-fold (Ta- Apparently, the aminoacylation reaction is limited by one or ble I). Elongation factor-1a activity, which was measured as a more of its reacting components. control, remained constant. 28916 Charging of Transfer RNA during Development of Artemia TABLE I developing embryos is largely due to the added (poly)ribosomal Aminoacyl-tRNA synthetase activities in extracts mRNAs rather than the endogenous mRNA. Together, these of quiescent and developing embryos observations confirm that the mRNA from developing embryos Aminoacyl-tRNA Specific activity is already loaded with ribosomes and translated as such by the synthetase Increase specific for Quiescent Developing quiescent embryo extract. Once the mRNA is initiated, the ribosomes elongate with a rate comparable to that in the de- units/mg units/mg -fold veloping embryo provided the restriction of charging of elon- Valine 43 67 1.6 gating tRNAs is released. Methionine 3.7 51 14 Lysine 180 400 2.2 It is known that phosphorylation is important in the regula- tion of the initiation step of protein synthesis (30), but so far Specific activities were calculated from the linear region of concen- tration-dependent plots obtained with extracts of quiescent and 20-h the evidence for phosphorylation playing an important role in developing embryo, incubated for 10 min at 25 °C in the presence of 3.5 synthetase function has been less conclusive, at least for ver- mg/ml yeast tRNA and H-labeled amino acid. One unit of activity tebrates (32). We prefer to think that a deficiency in the abso- corresponds to the formation of 1 pmol of aminoacyl-tRNA/min. lute amount of these enzymes may be responsible for the low synthetase activity in quiescent embryo extracts, as is also the DISCUSSION case in Bombyx mori (33). In fact, at least for ValRS, we have It is generally agreed that the rate-limiting steps in protein found by Western blotting that the amount of the protein synthesis lies at the level of initiation (30). The same situation increases significantly during development with retention of its does not necessarily prevail to reactivate protein synthesis intrinsic specificity. Since Northern blot analysis also reveal after a period of quiescence like in the brine shrimp Artemia. that the level of ValRS mRNA markedly increases during de- For instance, efforts to prove deficiencies in the initiation fac- velopment, the de novo synthesis of the ValRS enzyme may be tors of quiescent embryos have been rather unconvincing (31), attributed to enhanced transcription of the ValRS gene. It and levels of the initiation factor eIF-2 are found to be the same should be stressed that although the synthetase levels in qui- before and after development (10). Most characteristic of qui- escent embryos so far are substantial, apparently they are not escent embryos, however, is the inactive form of its mRNAs and high enough to recharge the pool of deacylated tRNAs within a the slow reappearance of polysomes during development (9, period of minutes, as in rapidly growing Escherichia coli and 13). However, this by itself does not explain that the encapsu- the re-activated spores of Bacillus megaterium (34). Synthetase lated embryo exhibits no protein synthesis unless resumption levels can never become zero in the quiescent embryo; they of development ensues. In this context, we have proven the should be low enough to prevent full charging, but high enough absence of an inhibitor, although adequate levels of mRNAs are to enable their own synthesis, because otherwise protein syn- present in such extracts (9). How protein synthesis is reacti- thesis would be irreversibly arrested. Careful studies on the vated during development of Artemia has therefore remained a transcriptional regulation of aminoacyl-tRNA synthetases dur- long-standing question (8 –15). We demonstrate that during ing development are therefore indicated. the development of quiescent embryos into metabolically active The aminoacylation of tRNA appears to function as a sensor embryos there is a dramatic increase in the level of tRNA that regulates the biosynthesis of amino acids via transcription aminoacylation. We also establish that the difference in protein factor GCN4 in yeast (35) and also the transcription of amino- synthesis capacity between extracts from quiescent and devel- acyl-tRNA synthetase genes in Gram-positive bacteria (36, 37). oping embryos can be completely abolished by solely adding We have demonstrated here that protein synthesis itself is fully charged aminoacyl-tRNA, together with the mixture of controlled at the level of tRNA charging during the natural polyribosomes of developing embryos as an external source of development of a eukaryote. Interestingly, in bacterial spores, translatable mRNA. Furthermore, we show by poly(U)-directed the level of tRNA charging is also very low and increases poly(Phe) synthesis that only the shortage of charged amino- rapidly upon germination (34). Moreover, tRNAs specific for acyl-tRNA restricts the elongation step of the quiescent em- valine, arginine, and histidine are among the most significantly bryo. In our opinion, our data prove for the first time that a charged tRNAs in these spores, as also seems to be the case in shortage of charged aminoacyl-tRNA in combination with a low Artemia embryos. Furthermore, studies on E. coli and mouse level of preformed polyribosomes is the cause of the extreme ascites tumor cells show that protein synthesis decreases when low rate of protein synthesis in extracts of the quiescent the charging degree of tRNAs is lowered on purpose (38, 39). embryo. Aminoacylation of tRNAs therefore appears to play a general The question which immediately comes to mind is what regulatory role in escaping routes from dormancy and may also regulates the charging of tRNAs in vivo during Artemia devel- be important for instance in germination of plant seeds and opment? Rather than addressing here how the charging level of perhaps also in the revival of G -arrested vertebrate cells. tRNAs have become low in the quiescent embryo, we focus on Since the present day adaptor molecule, tRNA, is believed to their reactivation after release from quiescence. Because we have been of prime importance in the genesis of protein syn- have shown that at least some of the aminoacyl-tRNA syn- thesis and life itself (40), the regulation of protein synthesis thetases, especially methionyl-tRNA synthetase, display an el- may even have started at the level of tRNA aminoacylation, evated activity in developing embryo lysates, they are prime while fine tuning of mRNA expression at the level of ribosome- candidates for the control of protein synthesis in Artemia. This induced initiation and elongation events occurred later on in immediately places the initiator tRNA in the limelight, and the evolution. 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