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Termination of Quiescence in Crustacea

1997; Elsevier BV; Volume: 272; Issue: 46 Linguagem: Inglês

10.1074/jbc.272.46.28912

1083-351X

Margreet Brandsma, George M. C. Janssen, W. Möller,

Crustacean biology and ecology

In quiescent embryos of the brine shrimpArtemia, the level of aminoacylation of transfer RNAs is low. During resumption of development the charging level of transfer RNAs increases, concomitant with the activation of protein synthesis. The total level of charging rises dramatically from an average of 4% to 50% within a period of 24 h of development. The restriction ofin vitro translation of the quiescent embryo extract can be partially released by the addition of chargedaminoacyl-tRNA, which apparently starts the flow of ribosomes into polyribosome structures. Complete reactivation of translation by aminoacyl-tRNA occurs when mRNA from preformed mRNA-ribosome complexes, like the polyribosomes extracted from developing embryos or poly(U)-programmed ribosomes, are offered to quiescent embryo extracts. With respect to the mechanism of in vivo recharging of tRNAs, we observed that the level of several aminoacyl-tRNA synthetases increase during development. Methionyl-tRNA synthetase rises more than 10-fold. In the case of valyl-tRNA synthetase, the activation is lower and shown to be due to the de novo synthesis of its mRNA and the corresponding protein product as well. We conclude that protein synthesis and thereby the gradual animation of cryptobiotic Artemia embryos is determined to a large extent by the rate by which aminoacyl-tRNAs are replenished during development at both the initiation andelongation level. In quiescent embryos of the brine shrimpArtemia, the level of aminoacylation of transfer RNAs is low. During resumption of development the charging level of transfer RNAs increases, concomitant with the activation of protein synthesis. The total level of charging rises dramatically from an average of 4% to 50% within a period of 24 h of development. The restriction ofin vitro translation of the quiescent embryo extract can be partially released by the addition of chargedaminoacyl-tRNA, which apparently starts the flow of ribosomes into polyribosome structures. Complete reactivation of translation by aminoacyl-tRNA occurs when mRNA from preformed mRNA-ribosome complexes, like the polyribosomes extracted from developing embryos or poly(U)-programmed ribosomes, are offered to quiescent embryo extracts. With respect to the mechanism of in vivo recharging of tRNAs, we observed that the level of several aminoacyl-tRNA synthetases increase during development. Methionyl-tRNA synthetase rises more than 10-fold. In the case of valyl-tRNA synthetase, the activation is lower and shown to be due to the de novo synthesis of its mRNA and the corresponding protein product as well. We conclude that protein synthesis and thereby the gradual animation of cryptobiotic Artemia embryos is determined to a large extent by the rate by which aminoacyl-tRNAs are replenished during development at both the initiation andelongation level. Multicellular organisms like the crustacean Artemia can survive long periods of environmental stress by entering a cryptobiotic stage (see Refs. 1Clegg J.S. Conte F.P. Persoone G. Sorgeloos P. Roels O. Jaspers E. The Brine Shrimp Artemia. 2. Universa Press, Wetteren, Belgium1980: 11-54Google Scholar, 2Lavens P. Sorgeloos P. Decleir W. Moens L. Slegers H. Sorgeloos P. Jaspers E. Artemia Research and Its Applications. 3. Universa Press, Wetteren, Belgium1987: 27-63Google Scholar, 3Crowe J.H. Crowe L.M. Drinkwater L. Busa W.B. Decleir W. Moens L. Slegers H. Sorgeloos P. Jaspers E. Artemia Research and Its Applications. 2. Universa Press, Wetteren, Belgium1987: 19-40Google Scholar, 4Clegg J.S. Drinkwater L.E. Sorgeloos P. Physiol. Zool. 1996; 69: 49-66Crossref Scopus (72) Google Scholar for reviews). Tissue culture cells can protect themselves against adverse growing conditions by entering the G0 phase of the cell cycle. In both cases, the rate of protein synthesis is reduced to a low level and its reactivation is a prerequisite for the cells to re-enter the cell cycle (5Golub A. Clegg J.S. Dev. Biol. 1968; 17: 644-656Crossref PubMed Scopus (37) Google Scholar, 6Thomas G. Gordon J. Cell Biol. Int. Rep. 1979; 3: 307-320Crossref PubMed Scopus (19) Google Scholar). For a better understanding of the processes that determine cell growth and division, it is important to know how protein synthesis can be regulated. Upon re-immersion of the quiescent dehydrated Artemia embryo in sea water, development quickly resumes. After a defined period of pre-emergence development, a free-swimming nauplius with distinct morphological features emerges from its shell. At the start of this period, protein synthesis and transcription resume quickly, both occurring in the absence of DNA synthesis or cell division (1Clegg J.S. Conte F.P. Persoone G. Sorgeloos P. Roels O. Jaspers E. The Brine Shrimp Artemia. 2. Universa Press, Wetteren, Belgium1980: 11-54Google Scholar, 7Möller W. Amons R. Janssen G. Lenstra J.A. Maassen J.A. Decleir W. Moens L. Slegers H. Sorgeloos P. Jaspers E. Artemia Research and Its Applications. 2. Universa Press, Wetteren, Belgium1987: 451-469Google Scholar). After 30 years of research on the re-activation of translation inArtemia, it is clear that quiescent and developing embryos contain approximately equal amounts of ribosomes (8Kenmochi N. Takahashi Y. Ogata K. J. Biochem. (Tokyo). 1989; 106: 289-293Crossref PubMed Scopus (4) Google Scholar), mRNA (9Grosfeld H. Littauer U.Z. Eur. J. Biochem. 1976; 70: 589-599Crossref PubMed Scopus (25) Google Scholar), initiation factors (10Macrae T.H. Roychowdhury M. Houston K.J. Woodley C.L. Wahba A.J. Eur. J. Biochem. 1979; 100: 67-76Crossref PubMed Scopus (21) Google Scholar), elongation factors (11.Janssen, G. M. C. (1994) Elongation Factor 1 from Artemia; Structure, Function and Its Regulation during Protein Synthesis. Ph.D. thesis, Leiden University, The Netherlands.Google Scholar), and termination factors (12Reddington M.A. Fong A.P. Tate W.P. Dev. Biol. 1978; 63: 402-411Crossref PubMed Scopus (8) Google Scholar), which are all equally active in in vitrotranslation assays. At the onset of development, a slow but definite shift from 80 S ribosomes to polysomes can be observed (5Golub A. Clegg J.S. Dev. Biol. 1968; 17: 644-656Crossref PubMed Scopus (37) Google Scholar, 13Hultin T. Morris J.E. Dev. Biol. 1968; 17: 143-164Crossref PubMed Scopus (41) Google Scholar). However, extracts from quiescent embryos are inactive in translation of their endogenous mRNAs (14Moreno A. Mendez R. De Haro C. Biochem. J. 1991; 276: 809-816Crossref PubMed Scopus (7) Google Scholar). This paradox has led several investigators to search for specific inhibitors and activators of translation in extracts from non-developing and developing embryos, respectively (15Lee-Huang S. Sierra J.M. Naranjo R. Filipowicz W. Ochoa S. Arch. Biochem. Biophys. 1977; 180: 276-287Crossref PubMed Scopus (48) Google Scholar). Until now, none of these approaches has provided a definite answer. Since ribosomes, factors, and mRNAs appear to be normal in the quiescent embryo, we have explored the level of charged tRNA during development. Although the amount of tRNA as such does not change significantly during development (16Bagshaw J.C. Finamore F.J. Novelli G.D. Dev. Biol. 1970; 23: 23-35Crossref PubMed Scopus (26) Google Scholar), the degree of tRNA aminoacylation has until now received little attention. The results of such studies are presented here. Dried Artemia embryos (San Francisco Bay Brand Inc., San Francisco, CA) were washed with 2% NaOCl as described (17Janssen G.M.C. Maassen J.A. Möller W. Spedding G. Ribosomes and Protein Synthesis. Oxford University Press, New York1990: 51-68Google Scholar) and either used directly (quiescent embryos) or cultured for the time indicated in aerated artificial sea water at 27 °C (developing embryos). Under these conditions, approximately 80% of the embryos emerge from their shells within 20 h. Dried embryos (25 g) were cultured 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 buffer) was added, and this mixture was homogenized three times for 45 s with a Polytron homogenizer. Upon centrifugation for 12 min at 3000 × g in a Beckman JA 10 rotor, the aqueous (upper) phase was extracted two more times with the same amount of acidic phenol. RNA from the final upper phase was precipitated with ethanol (18$$Google Scholar). The dried pellet was dissolved in 15 ml of 10 mm NaAc, pH 4.5, and lithium chloride was added to a concentration of 0.8 m, followed by centrifugation for 20 min at 9000 × g in a Sorvall SS 34 rotor. The tRNA present in the supernatant was reprecipitated with ethanol and then dissolved in 5 mm NaAc, pH 4.5, 0.5 m NaCl, and 10 mm MgCl2. Complete separation of tRNA from rRNA was achieved by gel filtration on a Superose 12 FPLC column in the same buffer. The tRNA eluting at the position of tRNA from brewers' yeast (Boehringer Mannheim) was pooled and reprecipitated with ethanol. The dried pellets were dissolved in 10 mm ammonium acetate, pH 4.5, and A 260 was measured. Samples containing 200 μg (3.7 A 260 units) of tRNA were adjusted to pH 8 by addition of ammonium carbonate, pH 8, to 100 mm and incubated for 1 h at 37 °C to achieve the complete deacylation of aminoacyl-tRNA (19Tockman J. Vold B.S. J. Bacteriol. 1977; 130: 1091-1097Crossref PubMed Google Scholar). Released amino acids were separated from tRNA by ultrafiltration (Microcon-3, Amicon), modified by dimethylaminoazobenzene sulfonyl chloride treatment, separated by reverse phase chromatography on a C18 high performance liquid chromatography column (100RP18e, Merck) and quantified at 436 nm by comparison to known amounts of amino acids (20Knecht R. Chang J. Wittmann-Liebold B. Salnikow J. Erdmann V.A. Advanced Methods in Protein Microsequence Analysis. Springer-Verlag, Berlin1986: 56-61Crossref Google Scholar). When the tRNA samples were incubated at pH 4.5 instead of 8, no significant amount of amino acid was found in the ultrafiltrate, indicating that our tRNA preparations are essentially devoid of contaminating free amino acids. Cell-free extracts from quiescent and developing Artemia embryos were prepared precisely as described in Ref. 14Moreno A. Mendez R. De Haro C. Biochem. J. 1991; 276: 809-816Crossref PubMed Scopus (7) Google Scholar. These extracts, referred to as “embryo lysate,” were used as such only for the in vitrotranslation experiments of Fig. 1. In the other experiments, 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, 1The abbreviations used are: DTE, dithioerythritol; ValRS, valyl-tRNA synthetase; poly(U), poly(uridylic acid). and 10% glycerol. Void-volume fractions, termed S30 extract, were pooled and stored in aliquots at −80 °C. The 100,000 × gsupernatant (S100) and 0.5 m KCl-washed (poly)ribosomal fraction as used in Fig. 4 were obtained from S30 extracts as described (17Janssen G.M.C. Maassen J.A. Möller W. Spedding G. Ribosomes and Protein Synthesis. Oxford University Press, New York1990: 51-68Google Scholar).Figure 4Reactivation of protein synthesis. A, in vitro protein synthesis on endogenous mRNAs by S30 extracts (30 μg of protein per 7 μl) of quiescent (○) and 20-h developing (•) embryos with the separate substrates, amino acids (35 μm each, except 10 μm[3H]Val) and uncharged tRNA (2.5 μg). B, the same as in A except that fully charged aminoacyl-tRNA (2.5 μg; labeled with [3H]Val) was used instead of the separate substrates. C, the same as in A except that S100 (17.5 μg of protein) was used instead of S30, together with the purified ribosomes (15 μg of protein) from developing embryos.D, the same as in C except that fully charged aminoacyl-tRNA (2.5 μg; labeled with [3H]Val) was used instead of the separate substrates. Since during S100 preparation the protein concentration decreased from ∼30 mg/ml in S30 to ∼17.5 mg/ml in S100, the amount of S100 protein was reduced proportionally. Assays were optimized with respect to the amount of ribosomes required. Initial rates were calculated from the data of the first 15 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 Mg2Cl, 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 400 μg of S100 protein from developing embryos and 200 μCi of [3H]Val. The aminoacyl-tRNAs were re-extracted with phenol as described (17Janssen G.M.C. Maassen J.A. Möller W. Spedding G. Ribosomes and Protein Synthesis. Oxford University Press, New York1990: 51-68Google Scholar) and purified by gel filtration on Superose 12. Judged from the [3H]Val incorporation of 1.09 nmol of Val/20.8 nmol of tRNA, each tRNA species was assumed to be fully charged with its cognate amino acid. The rate of in vitrotranslation was measured in an assay (40 μl) containing 20 mm Hepes, pH 7.6, 50 mm potassium acetate, 1.25 mm magnesium acetate, 0.1 mm spermidine, 0.2 mm DTE, 0.2 mm both GTP and ATP, 500 units/ml RNasin (Promega), 10 mm phosphocreatine, 25 units/ml creatine phosphokinase (Boehringer Mannheim), 3 μCi ofl-[3,4-3H]Val (specific radioactivity 10 Ci/mmol; Amersham), unlabeled l-amino acids (35 μm each, except Val), and extract as indicated in the legends for Figs. 1 and 4 (14Moreno A. Mendez R. De Haro C. Biochem. J. 1991; 276: 809-816Crossref PubMed Scopus (7) Google Scholar). Reactions were performed at 27 °C, and at indicated times, 7-μl aliquots were withdrawn, immediately added to 150 μl of ice-cold 10% (w/v) trichloroacetic acid, and heated for 15 min at 95 °C. The precipitate was collected on a glass fiber filter (GF/C; Whatman) and washed with three 2-ml aliquots of 10% trichloroacetic acid. The filter was dried at 95 °C (30 min) and its radioactivity measured in a liquid scintillation spectrometer. Conditions for globin synthesis were the same, except for the following. Globin mRNA (2.5 μg; Life Technologies, Inc.) was added to micrococcal nuclease-treated extracts (see below), and the complete reaction mixture (40 μl) was processed to determine the incorporation of [3H]Val into protein. Incorporation was found to be time-dependent and globin mRNA concentration-dependent with an optimum at 2.5 μg of globin mRNA. Poly(U)-directed poly(Phe) synthesis was adapted from (21Iwasaki K. Kaziro Y. Moldave K. RNA and Protein Synthesis. Academic Press, New York1981: 435-454Crossref Google Scholar). To remove the endogenous mRNA pool completely, S30 extracts (30 μl) were first treated for 15 min at 20 °C with 1 unit of micrococcal nuclease (Pharmacia) in the presence of 1 mm CaCl2. The nuclease was then inactivated by the addition of EGTA to a final concentration of 3 mm. Poly(Phe) synthesis was performed in a reaction mixture (40 μl) containing 20 mm Hepes, pH 7.6, 50 mmpotassium acetate, 1.25 mm magnesium acetate, 0.1 mm spermidine, 0.2 mm DTE, 0.2 mmboth GTP and ATP, 500 units/ml RNasin (Promega), 10 mmphosphocreatine, 25 units/ml creatine phosphokinase (Boehringer), 5A 260 units of poly(U)-programmed 80 S ribosomes from Artemia quiescent embryos (21Iwasaki K. Kaziro Y. Moldave K. RNA and Protein Synthesis. Academic Press, New York1981: 435-454Crossref Google Scholar), and nuclease-treated extract (20 μg of protein). The labeled compound was eitherArtemia [3H]Phe-tRNA (40 pmol; 850 cpm/pmol), previously labeled with l-[2,3,4,5,6-3H]Phe using extract from developing embryos as a source of phenylalanyl-tRNA synthetase and reisolated as described (17Janssen G.M.C. Maassen J.A. Möller W. Spedding G. Ribosomes and Protein Synthesis. Oxford University Press, New York1990: 51-68Google Scholar), or 3 μCi ofl-[2,3,4,5,6-3H]Phe (specific radioactivity 10 Ci/mmol, Amersham) together with deacylated tRNA from developingArtemia embryos (40 pmol). Reactions were performed at 27 °C, and 7-μl aliquots were withdrawn at specified times and processed as described above. Aminoacyl-tRNA synthetase activity was measured as described (22Brandsma M. Kerjan P. Dijk J. Janssen G.M.C. Möller W. Eur. J. Biochem. 1995; 233: 277-282Crossref PubMed Scopus (19) Google Scholar, 23Kellermann O. Viel C. Waller J.P. Eur. J. Biochem. 1978; 88: 197-204Crossref PubMed Scopus (26) Google Scholar). The amount of ValRS in extracts was analyzed by Western blotting, using an antiserum against Artemia ValRS (22Brandsma M. Kerjan P. Dijk J. Janssen G.M.C. Möller W. Eur. J. Biochem. 1995; 233: 277-282Crossref PubMed Scopus (19) Google Scholar, 24Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (47090) Google Scholar). The amount of ValRS mRNA was determined by Northern blotting, using a ValRS-specific cDNA probe, prepared by polymerase chain reaction and standard cloning techniques (18$$Google Scholar). Elongation factor-1α-dependent binding of [3H]Phe-tRNA to poly(U)-programmed 80 S ribosomes was performed as a control in the same extracts as described (17Janssen G.M.C. Maassen J.A. Möller W. Spedding G. Ribosomes and Protein Synthesis. Oxford University Press, New York1990: 51-68Google Scholar). Protein concentration was measured by the method of Lowry et al. (25Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The rate of in vitro translation of endogenous mRNAs is low in quiescent embryo extracts, but increases strongly throughout pre-emergence development (Fig.1 A). In Fig. 1 B, the time courses of [3H]Val incorporation into protein by extracts prepared from quiescent and 20-h developing embryos are compared. The latter exhibits a fairly steep incorporation throughout the whole incubation period, which is in excellent agreement with the results of De Haro's group, where [35S]Met was used as a label (14Moreno A. Mendez R. De Haro C. Biochem. J. 1991; 276: 809-816Crossref PubMed Scopus (7) Google Scholar). The incorporation of [3H]Phe and [3H]Leu into protein proceeds at a comparable rate. Concerning the nature of the process of reactivation of protein synthesis in Artemia, translational repressors and activators have been postulated to be present in quiescent and developing embryos, respectively (15Lee-Huang S. Sierra J.M. Naranjo R. Filipowicz W. Ochoa S. Arch. Biochem. Biophys. 1977; 180: 276-287Crossref PubMed Scopus (48) Google Scholar). However, when we mixed extracts prepared from quiescent and 20-h developing embryos in different ratios, we did not observe any effect of the extract from quiescent embryos on the translational activity of that of the developing embryos, nor the reverse. In fact, the extract prepared from quiescent embryos behaves as a buffer (Fig. 1 C). Thus, our results do not indicate the presence of a strong translational repressor in the quiescent nor an activator in the developing embryo, but rather suggest a shortage of one or several active translational components, which gradually get replenished during development of the quiescent embryo. We used direct amino acid analysis on purified aminoacyl-tRNA to determine the degree of in vivo aminoacylation. The purification procedure includes homogenization of embryos in phenol at pH 4.5 to prevent deacylation, isolation of total RNA, followed by precipitation with lithium chloride to remove the major part of rRNA (26Auffray C. Rougeon F. Eur. J. Biochem. 1980; 107: 303-314Crossref PubMed Scopus (2316) Google Scholar), and final purification of tRNA by gel filtration (see also Ref. 19Tockman J. Vold B.S. J. Bacteriol. 1977; 130: 1091-1097Crossref PubMed Google Scholar). Compared with the method of periodate oxidation, the major advantage of this procedure is that the charging degree of all 20 different tRNA species can be assessed in a single experiment. Neither the total yield of tRNA nor the ratio of tRNA to rRNA changed significantly during development. The average yield of 4.4 ± 0.8 mg (n = 6) tRNA/25 g of dried embryos is in the expected order when considering the EF-1α content of 22 mg/25 g of embryos (27Janssen G.M.C. van Damme H.T.F. Kriek J. Amons R. Möller W. J. Biol. Chem. 1994; 269: 31410-31417Abstract Full Text PDF PubMed Google Scholar) and assuming equal molar amounts of EF-1α and tRNA present in Artemia (28Moldave K. Annu. Rev. Biochem. 1985; 54: 1109-1149Crossref PubMed Scopus (405) Google Scholar). Moreover, the amino acid acceptance of tRNA prepared from quiescent and 20-h developing embryos was the same for valine, leucine, lysine, phenylalanine, and glycine, when using nauplius S100 as a source of aminoacyl-tRNA synthetases. In the case of valine, both tRNA pools could be charged to a maximum of 2.7% of the total tRNA pool by purified ValRS from Artemia (22Brandsma M. Kerjan P. Dijk J. Janssen G.M.C. Möller W. Eur. J. Biochem. 1995; 233: 277-282Crossref PubMed Scopus (19) Google Scholar). Under the assumption that tRNAVal represents 5% of the total tRNA pool, this indicates that at least half of the valine-accepting ends are intact, both in quiescent and developing embryos. We conclude that neither the total amount of tRNA nor its processing is significantly elevated during development. As seen from Fig. 2, there is a dramatic increase in the charging degree of each of the 20 tRNA-species during the first 20 h of development, from an average of 4 ± 4% (n = 3) in quiescent to 52 ± 3% (n = 2) in 20-h developing embryos. The mutual relationship between the charging degree of tRNA and the rate of translation is obvious, especially when taking into account that more than half of the 20 different tRNA species are not charged at all in the quiescent embryo (detection limit 2% charging). However, not all tRNAs appear to be recharged in concert. After 1 h, tRNAGlu already shows 40% charging, whereas tRNATrp after this time hardly carries any amino acid (Fig.2). Therefore, some aminoacyl-tRNA species may clearly contribute more to the repression of overall protein synthesis in quiescent embryo lysates than others. On the whole, the results of Figs. 1 and 2 clearly demonstrate a positive correlation between the capacity for protein synthesis and the degree of tRNA aminoacylation, and therefore support a model in which the arrest of translation in quiescentArtemia embryos is based on a shortage of its natural substrate, aminoacyl-tRNA. Whether the observed paucity of aminoacyl-tRNA actually restricts elongation can be determined experimentally by using the poly(U)-directed poly(phenylalanine) synthesis assay. The flow of [3H]phenylalanine into poly ([3H]phenylalanine) may be represented by two subsequent reactions (Reaction 1).[3H]Phe+tRNA⇄Phe-tRNA synthetase[3H]Phe-tRNA⇄elongation factorspoly([3H]Phe+tRNA)Reaction 1 When the substrates [3H]Phe and uncharged tRNA are added separately, the rate of poly(Phe) synthesis is expected to depend on the amounts of elongation factors and phenylalanyl-tRNA synthetase present in the extracts. In this case, the activity of the quiescent embryo extract is found to be about 2.5-fold lower than that of the developing embryo extract (Fig.3 A). However, when charged [3H]Phe-tRNA is used instead of [3H]Phe and tRNA, the difference in poly(Phe) synthesis between quiescent and developing embryo extracts disappears completely (Fig. 3 B). The results of Fig. 3 B indicate that elongation factors 1 and 2 are equally active in quiescent and developing embryos. We conclude that a shortage of charged phenylalanyl-tRNA limits the elongation phase of protein synthesis in extracts of quiescent embryos, at least under the direction of a synthetic messenger. The extent to which total cell-free protein synthesis of endogenous mRNA is limited by the shortage of aminoacyl-tRNAs was assessed as follows. In the presence of the two separate substrates, amino acids and total tRNA, the rate of protein synthesis in the quiescent embryo extract is about 16 times lower as that observed in the developing embryo, i.e. the relative rate is 6.4 ± 1.3% (mean ± S.D., n = 3) (Fig.4 A). Addition of the full complement of aminoacyl-tRNAs, however, markedly enhanced in vitro protein synthesis of the quiescent embryo from a relative rate of 6.4 ± 1.3% to a relative rate of 23.6 ± 1.2% (Fig. 4 B). This proves that a paucity of charged aminoacyl-tRNA significantly limits protein synthesis in extracts of the quiescent embryo. Since protein synthesis could not be further stimulated by up to 20 μm aminoacyl-tRNAs, a second restriction in translation of available mRNAs appears to be present in the quiescent embryo. Note that the level of mRNAs is comparable in both types of embryo extracts (9Grosfeld H. Littauer U.Z. Eur. J. Biochem. 1976; 70: 589-599Crossref PubMed Scopus (25) Google Scholar). As a first attempt, we fractionated the quiescent and developing embryo extracts into the cytosolic (S100), polyribosomal, and ribosomal wash fraction. Interestingly, the purified polyribosomal fraction of developing embryos was quite effective in stimulating protein synthesis of the quiescent embryo extract (S30), while the other fractions, including the ribosomes from quiescent embryos, were not. In fact, the polyribosomes from developing embryos activate protein synthesis of the quiescent embryo S100 to a relative rate 23.4 ± 1.6% of that of the developing embryo S100 (Fig.4 C). Ultimately, the further addition of fully charged aminoacyl-tRNAs completely restored protein synthesis in the extract of the quiescent embryo to the level of the developed embryo (Fig.4 D). Moreover, the ribosomal wash fractions, which contained large amounts of initiation factors (10Macrae T.H. Roychowdhury M. Houston K.J. Woodley C.L. Wahba A.J. Eur. J. Biochem. 1979; 100: 67-76Crossref PubMed Scopus (21) Google Scholar), were without effect, thus indicating that they were not rate-limiting. As shown previously by others, initiation factor eIF-2 was found to remain constant in amount and activity during development (10Macrae T.H. Roychowdhury M. Houston K.J. Woodley C.L. Wahba A.J. Eur. J. Biochem. 1979; 100: 67-76Crossref PubMed Scopus (21) Google Scholar). A substantial effect of charged tRNAs was also seen on the translation of globin mRNA by the quiescent embryo extract. 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 ± 8% to a relative rate of 76 ± 10%. Absolute values, however, were an order of magnitude less than those observed with polyribosomes in the two types of extract. This was also the case with translation of poly(A)+ mRNAs extracted from the developing embryo, indicating that “naked” mRNA is a poor substrate for translation by the quiescent and the developing embryo extract as well. We conclude, therefore, that during the development ofArtemia embryos, protein synthesis is activated by the recharging of aminoacyl-tRNAs, but due to the virtual absence of polyribosomes in the quiescent embryo (1Clegg J.S. Conte F.P. Persoone G. Sorgeloos P. Roels O. Jaspers E. The Brine Shrimp Artemia. 2. Universa Press, Wetteren, Belgium1980: 11-54Google Scholar, 5Golub A. Clegg J.S. Dev. Biol. 1968; 17: 644-656Crossref PubMed Scopus (37) Google Scholar, 13Hultin T. Morris J.E. Dev. Biol. 1968; 17: 143-164Crossref PubMed Scopus (41) Google Scholar), the rate of protein synthesis remains lower than in the developing embryo. Upon rehydration of the quiescent embryos, tRNA may be recharged by its cognate aminoacyl-tRNA synthetase as present at this stage. However, the low degree of charging of most of the tRNAs even after 1 h of development (Fig. 2) indicates a severe limitation in the supply of aminoacyl-tRNAs. Apparently, the aminoacylation reaction is limited by one or more of its reacting components.aa+tRNA+ATP⇄aminoacyl­tRNA synthetaseaa­tRNA+AMP+PPiREACTION2The tRNA pool of the quiescent embryo itself appears to be comparable to that of the developing embryo (this study and Ref. 16Bagshaw J.C. Finamore F.J. Novelli G.D. Dev. Biol. 1970; 23: 23-35Crossref PubMed Scopus (26) Google Scholar). The level of endogenous amino acids rises only about 2-fold (Ref. 1Clegg J.S. Conte F.P. Persoone G. Sorgeloos P. Roels O. Jaspers E. The Brine Shrimp Artemia. 2. Universa Press, Wetteren, Belgium1980: 11-54Google Scholar and our own observations), while the ATP concentration is cited to increase 7-fold during development (29Finnamore Clegg Padilla G.M. Whitson G.L. Cameron I.L. The Cell Cycle. Academic Press, New York1969: 249-278Crossref Google Scholar). However, the shortage of ATP by itself is not responsible for the inhibition of translation, since we found that the addition of ATP up to 1 mm does not restore protein synthesis in quiescent embryo extracts. Therefore, we investigated instead the behavior of three selected aminoacyl-tRNA synthetases, all of which were found to rise during the first 20 h of development, i.e. valyl- and lysyl-tRNA synthetase by a factor of about 2, while methionyl-tRNA synthetase increased more than 10-fold (Table I). Elongation factor-1α activity, which was measured as a control, remained constant.Table IAminoacyl-tRNA synthetase activities in extracts of quiescent and developing embryosAminoacyl-tRNA synthetase specific forSpecific activity1-aSpecific activities were calculated from the linear region of concentration-dependent plots obtained with extracts of quiescent and 20-h developing embryo, incubated for 10 min at 25 °C in the presence of 3.5 mg/ml yeast tRNA and 3H-labeled amino acid. One unit of activity corresponds to the formation of 1 pmol of aminoacyl-tRNA/min.IncreaseQuiescentDevelopingunits/mgunits/mg-foldValine43671.6Methionine3.75114Lysine1804002.21-a Specific activities were calculated from the linear region of concentration-dependent plots obtained with extracts of quiescent and 20-h developing embryo, incubated for 10 min at 25 °C in the presence of 3.5 mg/ml yeast tRNA and 3H-labeled amino acid. One unit of activity corresponds to the formation of 1 pmol of aminoacyl-tRNA/min. Open table in a new tab It is generally agreed that the rate-limiting steps in protein synthesis lies at the level of initiation (30Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (906) Google Scholar). The same situation does not necessarily prevail to reactivate protein synthesis after a period of quiescence like in the brine shrimp Artemia. For instance, efforts to prove deficiencies in the initiation factors of quiescent embryos have been rather unconvincing (31Sierra J.M. Meier D. Ochoa S. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2693-2697Crossref PubMed Scopus (41) Google Scholar), and levels of the initiation factor eIF-2 are found to be the same before and after development (10Macrae T.H. Roychowdhury M. Houston K.J. Woodley C.L. Wahba A.J. Eur. J. Biochem. 1979; 100: 67-76Crossref PubMed Scopus (21) Google Scholar). Most characteristic of quiescent embryos, however, is the inactive form of its mRNAs and the slow reappearance of polysomes during development (9Grosfeld H. Littauer U.Z. Eur. J. Biochem. 1976; 70: 589-599Crossref PubMed Scopus (25) Google Scholar, 13Hultin T. Morris J.E. Dev. Biol. 1968; 17: 143-164Crossref PubMed Scopus (41) Google Scholar). However, this by itself does not explain that the encapsulated embryo exhibits no protein synthesis unless resumption of development ensues. In this context, we have proven the absence of an inhibitor, although adequate levels of mRNAs are present in such extracts (9Grosfeld H. Littauer U.Z. Eur. J. Biochem. 1976; 70: 589-599Crossref PubMed Scopus (25) Google Scholar). How protein synthesis is reactivated during development of Artemia has therefore remained a long-standing question (8Kenmochi N. Takahashi Y. Ogata K. J. Biochem. (Tokyo). 1989; 106: 289-293Crossref PubMed Scopus (4) Google Scholar, 9Grosfeld H. Littauer U.Z. Eur. J. 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We demonstrate that during the development of quiescent embryos into metabolically active embryos there is a dramatic increase in the level of tRNA aminoacylation. We also establish that the difference in protein synthesis capacity between extracts from quiescent and developing embryos can be completely abolished by solely adding fully charged aminoacyl-tRNA, together with the mixture of polyribosomes of developing embryos as an external source of translatable mRNA. Furthermore, we show by poly(U)-directed poly(Phe) synthesis that only the shortage of charged aminoacyl-tRNA restricts the elongation step of the quiescent embryo. In our opinion, our data prove for the first time that a shortage of charged aminoacyl-tRNA in combination with a low level of preformed polyribosomes is the cause of the extreme low rate of protein synthesis in extracts of the quiescent embryo. The question which immediately comes to mind is what regulates the charging of tRNAs in vivo during Artemiadevelopment? Rather than addressing here how the charging level of tRNAs have become low in the quiescent embryo, we focus on their reactivation after release from quiescence. Because we have shown that at least some of the aminoacyl-tRNA synthetases, especially methionyl-tRNA synthetase, display an elevated activity in developing embryo lysates, they are prime candidates for the control of protein synthesis in Artemia. This immediately places the initiator tRNA in the limelight, and the shortage of charged initiator tRNAMet could explain the low fraction of ribosomes, present as polysomes in quiescent embryos (5Golub A. Clegg J.S. Dev. Biol. 1968; 17: 644-656Crossref PubMed Scopus (37) Google Scholar, 9Grosfeld H. Littauer U.Z. Eur. J. Biochem. 1976; 70: 589-599Crossref PubMed Scopus (25) Google Scholar, 13Hultin T. Morris J.E. Dev. Biol. 1968; 17: 143-164Crossref PubMed Scopus (41) Google Scholar). In fact, we have found that the polyribosomal fraction from developing embryos contains about 3 times more poly(A)+ RNA than that of quiescent embryos, in agreement with the observed shift from monosomes to polysomes at the onset of development (5Golub A. Clegg J.S. Dev. Biol. 1968; 17: 644-656Crossref PubMed Scopus (37) Google Scholar, 13Hultin T. Morris J.E. Dev. Biol. 1968; 17: 143-164Crossref PubMed Scopus (41) Google Scholar). Moreover, we found that the observed activation of the quiescent embryo extract by the (poly)ribosomal fraction from developing embryos is largely due to the added (poly)ribosomal mRNAs rather than the endogenous mRNA. Together, these observations confirm that the mRNA from developing embryos is already loaded with ribosomes and translated as such by the quiescent embryo extract. Once the mRNA is initiated, the ribosomes elongate with a rate comparable to that in the developing embryo provided the restriction of charging of elongating tRNAs is released. It is known that phosphorylation is important in the regulation of the initiation step of protein synthesis (30Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (906) Google Scholar), but so far the evidence for phosphorylation playing an important role in synthetase function has been less conclusive, at least for vertebrates (32Clemens M.J. Trends Biochem. Sci. 1990; 15: 172-175Abstract Full Text PDF PubMed Scopus (33) Google Scholar). We prefer to think that a deficiency in the absolute amount of these enzymes may be responsible for the low synthetase activity in quiescent embryo extracts, as is also the case in Bombyx mori (33Chang P.K. Dignam J.D. J. Biol. Chem. 1990; 265: 20898-20906Abstract Full Text PDF PubMed Google Scholar). In fact, at least for ValRS, we have found by Western blotting that the amount of the protein increases significantly during development with retention of its intrinsic specificity. Since Northern blot analysis also reveal that the level of ValRS mRNA markedly increases during development, the de novo synthesis of the ValRS enzyme may be attributed to enhanced transcription of the ValRS gene. It should be stressed that although the synthetase levels in quiescent embryos so far are substantial, apparently they are not high enough to recharge the pool of deacylated tRNAs within a period of minutes, as in rapidly growing Escherichia coli and the re-activated spores ofBacillus megaterium (34Setlow P. J. Bacteriol. 1974; 118: 1067-1074Crossref PubMed Google Scholar). Synthetase levels can never become zero in the quiescent embryo; they should be low enough to prevent full charging, but high enough to enable their own synthesis, because otherwise protein synthesis would be irreversibly arrested. Careful studies on the transcriptional regulation of aminoacyl-tRNA synthetases during development are therefore indicated. The aminoacylation of tRNA appears to function as a sensor that regulates the biosynthesis of amino acids via transcription factor GCN4 in yeast (35Lanker S. Bushman J.L. Hinnebusch A.G. Trachsel H. Meuller P.P. Cell. 1992; 70: 647-657Abstract Full Text PDF PubMed Scopus (46) Google Scholar) and also the transcription of aminoacyl-tRNA synthetase genes in Gram-positive bacteria (36Henkin T.A. Mol. Microbiol. 1994; 13: 381-387Crossref PubMed Scopus (84) Google Scholar, 37Putzer H. Laalami S. Brakhage A.A. Condon C Grunberg-Manago M. Mol. Microbiol. 1995; 16: 709-718Crossref PubMed Scopus (51) Google Scholar). We have demonstrated here that protein synthesis itself is controlled at the level of tRNA charging during the natural development of a eukaryote. Interestingly, in bacterial spores, the level of tRNA charging is also very low and increases rapidly upon germination (34Setlow P. J. Bacteriol. 1974; 118: 1067-1074Crossref PubMed Google Scholar). Moreover, tRNAs specific for valine, arginine, and histidine are among the most significantly charged tRNAs in these spores, as also seems to be the case inArtemia embryos. Furthermore, studies on E. coliand mouse ascites tumor cells show that protein synthesis decreases when the charging degree of tRNAs is lowered on purpose (38Rojiani M.V. Jakubowski H. Goldman E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1511-1515Crossref PubMed Scopus (28) Google Scholar, 39Ogilvie A. Huschka U. Kersten W. Biochim. Biophys. Acta. 1979; 565: 293-304Crossref PubMed Scopus (23) Google Scholar). Aminoacylation of tRNAs therefore appears to play a general regulatory role in escaping routes from dormancy and may also be important for instance in germination of plant seeds and perhaps also in the revival of G0-arrested vertebrate cells. Since the present day adaptor molecule, tRNA, is believed to have been of prime importance in the genesis of protein synthesis and life itself (40Crick F.H.C. J. Mol. Biol. 1968; 38: 367-379Crossref PubMed Scopus (1621) Google Scholar), the regulation of protein synthesis may even have started at the level of tRNA aminoacylation, while fine tuning of mRNA expression at the level of ribosome-induced initiation and elongation events occurred later on in evolution. We thank Dr. R. Amons and Dr. J. Dijk for their expert advice and Dr. B. Kraal and Dr. C. W. A. Pleij for critically reading the manuscript.