Starved Saccharomyces cerevisiae Cells Have the Capacity to Support Internal Initiation of Translation
1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês
10.1074/jbc.274.31.21741
ISSN1083-351X
AutoresIrit Paz, Lilach Abramovitz, Mordechai Choder,
Tópico(s)RNA Research and Splicing
ResumoInternal initiation of translation, whereby ribosomes are directed to internal AUG codon independently of the 5′ end of the mRNA, has been observed rarely in higher eucaryotes and has not been demonstrated in living yeast. We report here that starved yeast cells are capable of initiating translation of a dicistronic message internally. The studied element that functions as an internal ribosome entry site (IRES) is hardly functional or not functional at all in logarithmically growing cells. Moreover, during the logarithmic growth phase, this element seems to inhibit translation reinitiation when placed as an intercistronic spacer or to inhibit translation when placed in the 5′-untranslated region of a monocistronic message. Inhibition of translation is likely due to the putative strong secondary structure of the IRES that interferes with the cap-dependent scanning process. When cells exit the logarithmic growth phase, or when artificially starved for carbon source, translation of the IRES-containing messages is substantially induced. Our findings imply that the capacity to translate internally is a characteristic of starved rather than vegetatively growing yeast cells. Internal initiation of translation, whereby ribosomes are directed to internal AUG codon independently of the 5′ end of the mRNA, has been observed rarely in higher eucaryotes and has not been demonstrated in living yeast. We report here that starved yeast cells are capable of initiating translation of a dicistronic message internally. The studied element that functions as an internal ribosome entry site (IRES) is hardly functional or not functional at all in logarithmically growing cells. Moreover, during the logarithmic growth phase, this element seems to inhibit translation reinitiation when placed as an intercistronic spacer or to inhibit translation when placed in the 5′-untranslated region of a monocistronic message. Inhibition of translation is likely due to the putative strong secondary structure of the IRES that interferes with the cap-dependent scanning process. When cells exit the logarithmic growth phase, or when artificially starved for carbon source, translation of the IRES-containing messages is substantially induced. Our findings imply that the capacity to translate internally is a characteristic of starved rather than vegetatively growing yeast cells. The ribosome scanning model has been originally proposed by Kozak (1Kozak M. Annu. Rev. Cell Biol. 1992; 8: 197-225Crossref PubMed Scopus (416) Google Scholar) to explain how the translation process is initiated. Numerous studies have corroborated the model whereby the initiation complex is assembled near or at the 5′ end of the mRNA, facilitated by the interaction of the cap structure with the eucaryotic initiation factor 4E, and starts scanning the mRNA until the first AUG is encountered (for recent review see Ref. 2McCarthy J.E.G. Microbiol. Mol. Biol. Rev. 1998; 62: 1492-1553Crossref PubMed Google Scholar). An alternative mode of selecting an initiation codon, whereby ribosomes are directed to an internal AUG by an internal ribosome entry sequence (IRES), 1The abbreviations used are: IRES, internal ribosome entry sequence; SP, stationary phase; SP1, 1 day in SP; SP4, 4 days in SP; SG, slow growth phase; UTR, untranslated region; GFP, green fluorescent protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; βgal, β-galactosidase; bp, base pair(s); ORF, open reading frame. has also been demonstrated. Well documented cases of internal initiation events are those of the uncapped picornaviral mRNAs (3Ehrenfeld E. Hershey J.W.B. Mathews M.B. Sonenberg N. Translation Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 549-573Google Scholar). IRESes have also been found in the 5′-untranslated region (5′-UTR) of several cellular mRNAs (4Sarnow P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5795-5799Crossref PubMed Scopus (87) Google Scholar, 5Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (422) Google Scholar, 6Oh S.K. Scott M.P. Sarnow P. Genes Dev. 1992; 6: 1643-1653Crossref PubMed Scopus (169) Google Scholar, 7Vagner S. Gensac M.C. Maret A. Bayard F. Amalric F. Prats H. Prats A.C. Mol. Cell. Biol. 1995; 15: 35-44Crossref PubMed Scopus (286) Google Scholar, 8Gan W. Rhoads R.E. J. Biol. Chem. 1996; 271: 623-626Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Vagner S. Touriol C. Galy B. Audigier S. Gensac M.C. Amalric F. Bayard F. Prats H. Prats A.C. J. Cell Biol. 1996; 135: 1391-1402Crossref PubMed Scopus (128) Google Scholar, 10Bernstein J. Sella O. Le S.Y. Elroy-Stein O. J. Biol. Chem. 1997; 272: 9356-9362Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Nanbru C. Lafon I. Audigier S. Gensac M.C. Vagner S. Huez G. Prats A.C. J. Biol. Chem. 1997; 272: 32061-32066Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Ye X. Fong P. Iizuka N. Choate D. Cavener D.R. Mol. Cell. Biol. 1997; 17: 1714-1721Crossref PubMed Scopus (78) Google Scholar, 13Gan W. Celle M.L. Rhoads R.E. J. Biol. Chem. 1998; 273: 5006-5012Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). During evolution, IRESes have been utilized as targets for translation regulation during normal differentiation and development. For example, an IRES was shown to play a role in the translation of platelet-derived growth factor 2 mRNA that increases after megacaryocitic cells undergo terminal differentiation (10Bernstein J. Sella O. Le S.Y. Elroy-Stein O. J. Biol. Chem. 1997; 272: 9356-9362Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). IRESes have also been found to mediate the differential translation of Antenapedia and Ultrabithorax during Drosophila melanogaster development (6Oh S.K. Scott M.P. Sarnow P. Genes Dev. 1992; 6: 1643-1653Crossref PubMed Scopus (169) Google Scholar, 12Ye X. Fong P. Iizuka N. Choate D. Cavener D.R. Mol. Cell. Biol. 1997; 17: 1714-1721Crossref PubMed Scopus (78) Google Scholar). Surprisingly, no IRES has been shown to function in yeasts, despite the observations that the yeast cell-free system is capable of recognizing plant viral IRESes (14Altmann M. Blum S. Wilson T.M. Trachsel H. Gene. 1990; 91: 127-129Crossref PubMed Scopus (35) Google Scholar) as well as natural yeast leader sequences (15Iizuka N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar). Attempts to promote internal initiation in living yeast cells by using IRESes of poliovirus (16Coward P. Dasgupta A. J. Virol. 1992; 66: 286-295Crossref PubMed Google Scholar) and encephalomyocarditis virus (17Evstafieva A.G. Beletsky A.V. Borovjagin A.V. Bogdanov A.A. FEBS Lett. 1993; 335: 273-276Crossref PubMed Scopus (20) Google Scholar) have thus far failed. These studies were done with optimally growing cells. In their natural environment, however, yeast occasionally encounter starvation and enter into a distinct quiescent state called stationary phase (SP) (reviewed in Refs. 18Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar and 19Werner-Washburne M. Braun E.L. Crawford M.E. Peck V.M. Mol. Microbiol. 1996; 19: 1159-1166Crossref PubMed Scopus (183) Google Scholar). In the test tube, when yeast cells are cultured in rich media (e.g. YPD), nutrient consumption is gradual and entry into SP occurs in a stepwise manner. Thus, cells cultured in YPD display a characteristic growth pattern in which log phase is followed by a diauxic shift, a slow growth phase, and then by SP. During the diauxic shift, cell metabolism changes from mainly fermentation to aerobic metabolism (20Entian K.D. Microbiol. Sci. 1986; 3: 366-371PubMed Google Scholar), accompanied by morphological and biochemical changes (21Boucherie H. J. Bacteriol. 1985; 161: 385-392Crossref PubMed Google Scholar, 22Boy-Marcotte E. Tadi D. Perrot M. Boucherie H. Jacquet M. Microbiology. 1996; 142: 459-467Crossref PubMed Scopus (63) Google Scholar, 23Costa V. Amorim M.A. Reis E. Quintanilha A. Moradas-Ferreira P. Microbiology. 1997; 143: 1649-1656Crossref PubMed Scopus (103) Google Scholar, 24Rubbi L. Camilloni G. Caserta M. Di Mauro E. Venditti S. Biochem. J. 1997; 328: 401-407Crossref PubMed Scopus (8) Google Scholar, 25Boy-Marcotte E. Perrot M. Bussereau F. Boucherie H. Jacquet M. J. Bacteriol. 1998; 180: 1044-1052Crossref PubMed Google Scholar). To preserve energy during SP, yeast cells shut off the transcription of most genes. Consequently, the global mRNA level is reduced about 35-fold (27Choder M. Genes Dev. 1991; 5: 2315-2326Crossref PubMed Scopus (98) Google Scholar,28Choder M. Young R.A. Mol. Cell. Biol. 1993; 13: 6984-6991Crossref PubMed Scopus (115) Google Scholar). Protein production during SP is reduced even more dramatically (200–300-fold) (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar), suggesting that the availability of mRNAs is not the rate-limiting factor of protein synthesis. Instead, translation during SP is found to be inefficient or even actively repressed. Nevertheless, expression of a small repertoire of genes is not repressed in SP (18Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar, 19Werner-Washburne M. Braun E.L. Crawford M.E. Peck V.M. Mol. Microbiol. 1996; 19: 1159-1166Crossref PubMed Scopus (183) Google Scholar, 27Choder M. Genes Dev. 1991; 5: 2315-2326Crossref PubMed Scopus (98) Google Scholar, 29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar). It is quite possible that the control of this dramatic strategic change in gene expression requires SP-specific mechanisms for ensuring the synthesis of the small repertoire of proteins that are essential for life during starvation. Here we show that yeast cells are capable of recognizing an IRES element. The RNA element that functions as an IRES was found by serendipity and contains a sequence from the Escherichia coli lacI. Strikingly, the capacity to recognize this IRES is found in starved cells but not in growing cells. We therefore call this IRESstationary phase-induced IRES, SIRES. By adapting a colony color test, we utilized the ACT1p-UBI4-lacZ reporter, described under "Results," and searched for mutants that failed to repress the reporter gene during SP. We thus identified a cell line whose reporter produced high levels of βgal in SP. Later, we discovered that the defect was not in the cellular genome but, instead, in the plasmid that underwent a rearrangement event. A series of experiments have demonstrated that the RNA element, which was responsible for the translational induction in SP, contains the sequence shown in Fig. 1B. Constructs are schematized in Fig. 1A. Construct 1 was described previously (26Meunier J.-R. Choder M. Yeast. 1999; (in press)PubMed Google Scholar). Construct 2 was made by inserting SIRES at the HindIII site, located in-between ACT1p and UBI4, into construct 1. For construct 3, green fluorescent protein (GFP) was amplified by PCR reaction, using OMC116 (5′-CCCAAGCTTGGATCCTAAAGATGA GTAAAGGAG-3′) and OMC117 (5′-CCCAAGCTTTCTAGATTATCATTCATCCATGCCATGTG-3′3′) as the forward and reverse primers, respectively.HindIII sites are underlined and were used to introduce the PCR fragment into HindIII site of construct 1 (betweenACT1p and UBI4). Two in-frame stop codons (marked by bold letters) are introduced immediately after the last codon of the GFP. XbaI was introduced downstream of theHindIII site and was used for inserting the intercistronic spacers of constructs 4–6. For constructs 4–6, intercistronic spacers were amplified by PCR technology, using primers that contained theXbaI site at their 5′ ends. The spacers were introduced in the single XbaI site of construct 3. All constructs were subject to sequencing analysis. Cell extract was equilibrated with Laemmli sample buffer and kept at room temperature. Ten μg were subjected to SDS-PAGE analysis. Fluorescent bands were detected by exposing the gel to Image reader FLA2000 (Fujifilm) at 473 nm excitation and using the 520 nm filter. SIRES has been discovered by serendipity during a screen for SP-specific mutations (see "Experimental Procedures"). It contains a sequence from the E. coli lacIgene and a portion of a multiple cloning region. The sequence does not contain AUG or other codons that can function as translation start sites (Fig. 1B). It contains a high GC content (62%) and, by subjecting its sequence to a folding algorithm (foldRNA, GCG Sequence Analysis Package), was found to be capable of forming a highly structured molecule (ΔG = −43.8 kcal mol−1). To study the effect of SIRES on the translation we used the UBI4-lacZ, encoding a ubiquitin-βgal fusion that contains isoleucine at the junction between the two protein sequences. Shortly after translation, the ubiquitin is cleaved off the fusion protein, and the resulting βgal is short lived both in dividing (30Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1383) Google Scholar) and in stationary cells (39Paz I. Meunier J.-R. Choder M. Gene. 1999; (in press)PubMed Google Scholar). This reporter thus enables the coupling between the level of the unstable βgal and the rate of its synthesis, even when this rate is decreased (39Paz I. Meunier J.-R. Choder M. Gene. 1999; (in press)PubMed Google Scholar). Transcription of the reporter gene is governed by ACT1promoter fragment, spanning positions +1 to −472 relative to the firstACT1 ATG codon and covering the previously described regulatory elements and transcription start sites of the gene (32Munholland J.M. Kelly J.K. Wildeman A.G. Nucleic Acids Res. 1990; 18: 6061-6068Crossref PubMed Scopus (16) Google Scholar) (construct 1 in Fig. 1A). SIRES was introduced downstream of the ACT1p and upstream of the UBI4 translation start codon in the ACT1p-UBI4-lacZ, as schematically shown in Figs. 1A and 2A,right panels (see also "Experimental Procedures"). The inclusion of SIRES had little effect on the level of the ACT1p-UBI4-lacZ transcript (Fig. 2B, compareleft and right panels). In contrast, the presence of SIRES had a dramatic effect on the level of the translated protein, shown in Fig. 2C (compare leftand right panels). During the logarithmic growth phase, the βgal level in cells carrying the SIRES-containing construct was two orders of magnitude lower than that in cells carrying the control construct (log lanes in Fig. 2C, compare theright and left panels). Thus, in logarithmically growing cells, the presence of SIRES in the 5′-UTR inhibits translation. This result is consistent with previous observations demonstrating that in yeast the inclusion in a 5′-UTR of an element that has the potential to form a secondary structure with a stability of ≥28 kcal mol−1 inhibits translation by at least 98% (33Vega Laso M.R. Zhu D. Sagliocco F. Brown A.J. Tuite M.F. McCarthy J.E. J. Biol. Chem. 1993; 268: 6453-6462Abstract Full Text PDF PubMed Google Scholar). However, other explanations for the SIRES-mediated translational repression cannot be ruled out. For example SIRES may give the mRNA a competitive disadvantage. The differential translation of the control mRNA and the SIRES-containing mRNA during log phase, when the translation of the former was higher than that of the latter, underwent a significant twist after the diauxic shift. Specifically, expression ofACT1p-UBI4-lacZ was repressed as cells exited the log phase (see above; Fig. 2, left panels). This result represents the general repression of translation characteristic of starved cells (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar, 34Martinez-Pastor M.T. Estruch F. FEBS Lett. 1996; 390: 319-322Crossref PubMed Scopus (33) Google Scholar, 39Paz I. Meunier J.-R. Choder M. Gene. 1999; (in press)PubMed Google Scholar). In contrast, expression of ACT1p-SIRES-UBI4-lacZwas induced after the diauxic shift, as demonstrated by the increase in the βgal product and activity (Fig. 2C, right panels). Note that, in post-log phases, the translation of the SIRES-containing mRNA was higher than the translation of the control mRNA, suggesting that the induction was due to activation rather than derepression. Interestingly, the increase in βgal level and activity occurred concomitantly with a decrease in the mRNA level (compare the changes in the mRNA levels in Fig. 2B, right panel, with those of the protein in Fig. 2C, right panel), indicating that the translatability of the SIRES-containing mRNA enhanced more substantially than the observed increase of the protein. Remarkably, the enhanced translation of the SIRES-containing mRNA occurred concomitantly with the general repression of translation. Attempts to identify the environmental stimuli that lead to the SIRES-mediated translational induction revealed that depletion of the sugar resulted in a specific translational enhancement of the SIRES-containing mRNA (results not shown). Two observations raised the possibility that SIRES mediates cap-independent translation. First, SIRES is capable of forming a highly stable secondary structure, which is likely to impede the cap-dependent ribosome scanning process (see above). Second, SIRES-mediated translation is enhanced when the bulk of the translation machinery is compromised, raising the possibility that it is recognized by a different or modified machinery. To study the mode of action of SIRES, a series of dicistronic constructs were made (Fig. 1A). Dicistronic constructs have been effectively usedin vivo to demonstrate the existence of IRES (3Ehrenfeld E. Hershey J.W.B. Mathews M.B. Sonenberg N. Translation Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 549-573Google Scholar, 5Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (422) Google Scholar, 6Oh S.K. Scott M.P. Sarnow P. Genes Dev. 1992; 6: 1643-1653Crossref PubMed Scopus (169) Google Scholar, 8Gan W. Rhoads R.E. J. Biol. Chem. 1996; 271: 623-626Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,13Gan W. Celle M.L. Rhoads R.E. J. Biol. Chem. 1998; 273: 5006-5012Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). As most ribosomes fail to continue through the intercistronic spacer, the translation of the second cistron is greatly reduced, unless preceded by an IRES. Using these constructs we attempted to answer the following questions. (i) Can SIRES promote translation of the second cistron by internal initiation? (ii) Is the translation of the second cistron influenced by that of the first cistron, or is the translation of the two cistrons controlled independently? (iii) Is the translation of the second cistron induced in SP like that of the SIRES-containing monocistronic mRNA? The gene encoding the GFP was chosen as the first cistron in the dicistronic constructs, and the second cistron wasUBI4-lacZ. (Fig. 1A). To enhance translational termination of the first cistron and to minimize reinitiation of the second one due to "leakiness" from the first cistron, we introduced an additional stop codon immediately downstream to the natural GFP stop codon. SIRES was placed as the intercistronic region, downstream to the stop codons. Three SIRES-less constructs were engineered to be used as controls. One control construct contained no intercistronic region (except for two restriction sites of 12 bp used for the constructions). Two other control sequences were introduced in lieu of SIRES; a 104-bp sequence (from the 5′-noncoding region ofUBI4) or a 282-bp sequence (from the 5′-noncoding region ofHAP4). To determine the effect of SIRES on the translation of both cistrons, equal amounts of protein, produced by the transformants with the different constructs, were separated by SDS-PAGE; the GFP level was assayed by monitoring its fluorescent intensity, and the βgal level was determined by Western analysis. Fig. 3B (lanes 14–17) shows that the level of βgal produced by the SIRES-containing dicistronic mRNA was almost undetectable in log phase cells and substantially increased in post-log phases. The induced synthesis of βgal in post-log cells occurred concomitantly with the observed decrease in the mRNA level (see below), indicating that the translatability of the SIRES-containing mRNA in post-log phases was enhanced even more than can be deduced from the observed increase of the protein. In contrast to translation of the second cistron, translation of the first cistron was high in log phase and lower in post-log phases (Fig. 3A, lanes 14–17). We suppose that the decrease of GFP translation in post-log phases is stronger than the observed decrease in its steady state level because GFP, like most proteins (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar), is highly stable in SP. The different expression behavior of the two cistrons indicates that, after the diauxic shift, the translation of the two cistrons is independently controlled. The expression pattern of the SIRES-containing construct was compared with that of the control constructs. In cells carrying the spacerless dicistronic mRNA no βgal was detected, indicating that following termination of the GFP translation no reinitiation occurred. It was demonstrated previously that reinitiation can occur in yeast and that the spacing between the sites of termination and initiation is important (2McCarthy J.E.G. Microbiol. Mol. Biol. Rev. 1998; 62: 1492-1553Crossref PubMed Google Scholar). We therefore examined expression of the second cistron in cells carrying the other control constructs. Indeed, the inclusion of either a 104- or a 282-nucleotide spacer in the dicistronic mRNA permitted little synthesis of βgal during log phase, which was detected only after overexposure (lanes 6 and 10 in Fig. 3B, lower panel). This βgal synthesis is most probably the result of translational reinitiation. In post-log phases, the level of the βgal expressed by these control dicistronic mRNAs declined (lanes 7–9 and 11–13 in Fig. 3B, lower panel). This is in contrast with the increase in the level of βgal expressed by the SIRES-containing mRNA (lanes 15–17 in Fig. 3B). In summary, the differences between the expression pattern of the control constructs and the expression pattern of the SIRES-containing construct indicates that the induced synthesis of the second cistron is SIRES-specific. We consider the possibility that the SIRES-mediated translation of the second cistron is due to reinitiation as unlikely because it is much higher than the translation of the second cistron of the control mRNAs. Furthermore, the observation that following the diauxic shift the translation of the two cistrons that are separated by SIRES is independently controlled (i.e. one is repressed and the other one is induced) strongly argues against the reinitiation possibility. Interestingly, the level of βgal produced by the SIRES-containing mRNA during log phase was even lower than that produced by the mRNA containing the control spacers (compare lane 14with lanes 6 and 10, in Fig. 3B,lower panel). This result is consistent with our suggestion that SIRES, which is not active as IRES in log phase, impedes the scanning-mediated reinitiation process. We then examined whether the SIRES-containing construct gave rise to one transcript that encompasses both GFP and UBI4-lacZ genes. Transcripts were analyzed by Northern blot hybridization using either GFP or lacZ as probes. Fig. 4 shows that the SIRES-containing dicistronic gene produced a dicistronic transcript, which migrated slower than the monocistronic transcript (comparelane 1 with lanes 2–5) and hybridized with both probes. The dicistronic message was by far the most prominent RNA species that was detected by both probes. In addition to this band, a few minor bands were detected after overexposure. These RNA species could not have promoted translation of the second cistron by an end-initiated scanning process for the following reasons: (i) no RNA species was detected that hybridized only (or preferentially) withlacZ but not with the GFP probe, indicating that no transcript has a 5′ end located downstream of the GFP open reading frame that might promote a cap-dependent translation of the second cistron, and (ii) the intensities of the minor bands were not increased after the shift from log to post-log phases. Because SIRES mediates an increase in the expression of the second cistron in post-log phases (see Fig. 3), this increase cannot be attributed to one of the minor bands. We conclude that the cells can support an induced translation of the second cistron during post-log phases only by internal initiation of translation (see also the note in the legend to Fig. 4). Taken together, the results shown in Figs. 3 and 4 indicate that translation of the two cistrons that are separated by SIRES is controlled differently. Thus, following the shift from log to post-log phases, when the translation of the first cistron decreases, translation of the second cistron increases. We conclude that, after cells exit the log phase, SIRES promotes the initiation of the second cistron translation from an internal site in the mRNA. Little or no internal initiation can be detected in growing cells. Previous experiments failed to identify IRESes in vegetatively growing yeast (see introduction). The lack of success of finding IRESes in dividing cells, taken together with our results that the capacity to recognize an IRES is found in starved cells, raises the possibility that putative IRESes are not recognized in optimally growing yeast cells but rather in starved, or otherwise stressed, cells. Cumulative data from several laboratories suggest that IRESes are best recognized when the main initiation pathway is compromised. The most remarkable examples are the observations that the activities of picornaviral IRESes (3Ehrenfeld E. Hershey J.W.B. Mathews M.B. Sonenberg N. Translation Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 549-573Google Scholar) and BiP IRES (4Sarnow P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5795-5799Crossref PubMed Scopus (87) Google Scholar, 5Macejak D.G. Sarnow P. Nature. 1991; 353: 90-94Crossref PubMed Scopus (422) Google Scholar) are enhanced as a result of the inactivation of the cap-dependent mechanism. Similarly, when yeast encounter starvation, the overall translation declines by more than 2 orders of magnitude (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar, 34Martinez-Pastor M.T. Estruch F. FEBS Lett. 1996; 390: 319-322Crossref PubMed Scopus (33) Google Scholar). We propose that a capacity to recognize putative natural SIRESes has evolved in yeast to provide a means to escape the general loss of the cap-dependent translation capacity, and that SIRES is fortuitously recognized by this machinery. Thus, putative natural SIRESes are likely to render the translation of some mRNAs, encoding proteins that are important for surviving starvation, independent of the main initiation pathway. SIRES can be utilized in the future to isolate factors that are capable of identifying IRESes during starvation. Identifying these factors may also help with the identification of natural SIRESes. It has been found that a yeast cell-free system, prepared from logarithmically growing Saccharomyces cerevisiae cells, can recognize heterologous authentic IRESes (14Altmann M. Blum S. Wilson T.M. Trachsel H. Gene. 1990; 91: 127-129Crossref PubMed Scopus (35) Google Scholar, 15Iizuka N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar) or yeast UTR sequences as IRESes (15Iizuka N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar). However, natural IRESes have not been identified in living yeast cells. This discrepancy suggests that, for the identification of IRESes, the currently available in vitrosystems might be irrelevant to the biological systems. Alternatively, it is possible that the capacity to recognize IRESes exists also in log phase yeast cells, and the in vitro system reliably detects this capacity. However, this capacity is repressed in dividing cells, either because the cap-recognizing machinery competes very efficiently with the IRES-recognizing machinery or because dividing cells express specific repressor(s), or because of both possibilities. According to this view, one or more of these repressing features is lost in thein vitro system. Starvation-induced increase of translation has been previously demonstrated in the case of GCN4 (review in Ref. 35Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). In a series of studies, Hinnebusch and his co-workers (35Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) have shown that induction of GCN4 translation in response to amino acid or purine deprivation is mediated by four short open reading frames (uORFs) in the leader of its mRNA. The uORFs inhibit theGCN4 translation in nonstarved cells by restricting the progression of the scanning ribosomes through the leader. Upon starvation, the scanning ribosomes bypass the most inhibitory uORF, uORF4, and repression is partially relieved, leading to an increased translation of the GCN4 ORF (35Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Apparently, the starvation-induced up-regulation of GCN4 differs from that mediated by SIRES. First, up-regulation of GCN4 translation results from a derepression mechanism that is imposed by the uORFs, whereas the SIRES-mediated translation seems to be governed by activation. Second, up-regulation of GCN4 mRNA translation is cap-dependent, whereas that mediated by SIRES is not. We propose that during starvation, when the general cap-dependent translation is repressed (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar, 34Martinez-Pastor M.T. Estruch F. FEBS Lett. 1996; 390: 319-322Crossref PubMed Scopus (33) Google Scholar), there are at least two types of mechanisms that mediate the translation of a small repertoire of mRNAs whose products are important for coping with starvation. We would like to thank O. Elroy-Stein, S. Ben-Yehuda, and N. Koleteva-Levine for critically reading the manuscript and A. Varshavsky and A. Sentenac for plasmids and antibodies. We are grateful to all members of Dr. Choder's laboratory for discussions, advice, and helpful suggestions.
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