Starved Saccharomyces cerevisiae Cells Have the Capacity to Support Internal Initiation of Translation

Abstract

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. 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 I. J.-R. Choder M. Gene. 1999; (in press)PubMed Google Scholar). reporter thus the between the level of the βgal and the of its even when this is I. J.-R. Choder M. Gene. 1999; (in press)PubMed Google Scholar). of the reporter gene is by to to the codon and the previously described and transcription start sites of the gene A.G. 1990; PubMed Scopus Google Scholar) 1 in Fig. SIRES was introduced downstream of the ACT1p and of the translation start codon in the as shown in and (see also “Experimental Procedures”). The of SIRES effect on the level of the ACT1p-UBI4-lacZ (Fig. and In the of SIRES a dramatic effect on the level of the protein, shown in Fig. During the logarithmic growth phase, the βgal level in cells the construct was two of than that in cells the control construct in Fig. and Thus, in logarithmically growing the of SIRES in the is with observations that in yeast the in a of an element that has the to a secondary structure with a of kcal translation by at D. F. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). other for the translational be For SIRES the mRNA a The differential translation of the control mRNA and the mRNA during log phase, when the translation of the was higher than that of the underwent a after the diauxic expression was repressed as cells the log phase (see Fig. the 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, F. FEBS Lett. 1996; PubMed Scopus Google Scholar, I. J.-R. Choder M. Gene. 1999; (in press)PubMed Google Scholar). In expression of after the diauxic shift, as demonstrated by the in the βgal and (Fig. in the translation of the mRNA was higher than the translation of the control mRNA, suggesting that the induction was due to rather than the in βgal level and with a in the mRNA level the changes in the mRNA levels in Fig. with those of the protein in Fig. that the of the mRNA more substantially than the observed of the the translation of the mRNA with the of Attempts to the that to the translational induction that of the in a translational of the mRNA not Two observations the that SIRES SIRES is capable of forming a highly secondary which is likely to the cap-dependent ribosome scanning process (see translation is when the of the translation is the that is by a or To study the mode of of a series of dicistronic constructs were made (Fig. constructs have been to the 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 W. Celle M.L. Rhoads R.E. J. Biol. Chem. 1998; 273: 5006-5012Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). most ribosomes to the intercistronic the translation of the is by an constructs we to the SIRES promote translation of the by internal the translation of the by that of the first or is the translation of the two the translation of the in SP that of the monocistronic The gene encoding the was as the first in the dicistronic and the (Fig. To translational of the first and to reinitiation of the due to from the first we introduced an stop codon immediately downstream to the natural stop SIRES was placed as the intercistronic downstream to the stop constructs were to be used as control construct contained no intercistronic region for two sites of used for the Two other control sequences were introduced in of a sequence the region or a sequence the region To the effect of SIRES on the translation of both of protein, produced by the with the were by the level was by its fluorescent and the βgal level was by analysis. Fig. that the level of βgal produced by the dicistronic mRNA was in log phase cells and substantially in The synthesis of βgal in cells with the observed in the mRNA level (see that the of the mRNA in was even more than can be from the observed of the In to translation of the translation of the first was high in log phase and in (Fig. We that the of translation in is than the observed in its state level GFP, most proteins (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar), is highly in SP. The expression of the two after the diauxic shift, the translation of the two is independently The expression pattern of the construct was with that of the control In cells the dicistronic mRNA no βgal was that of the translation no reinitiation It was demonstrated previously that reinitiation can in yeast and that the between the sites of and initiation is J.E.G. Microbiol. Mol. Biol. Rev. 1998; 62: 1492-1553Crossref PubMed Google Scholar). We therefore expression of the in cells the other control the of a or a spacer in the dicistronic mRNA synthesis of βgal during log phase, which was detected after and in Fig. βgal synthesis is most the of translational In the level of the βgal by control dicistronic mRNAs and in Fig. is in with the in the level of βgal by the mRNA in Fig. In the between the expression pattern of the control constructs and the expression pattern of the construct that the synthesis of the is We the that the translation of the is due to reinitiation as is higher than the translation of the of the control the that the diauxic the translation of the two that are by SIRES is independently is repressed and the other is the reinitiation the level of βgal produced by the mRNA during log phase was even than that produced by the mRNA the control spacers and in Fig. is with that which is not as IRES in log phase, the reinitiation process. We then the construct to that both and genes. were by using or as Fig. 4 that the dicistronic gene produced a dicistronic which than the monocistronic 1 with and with both The dicistronic message was by far the most RNA that was detected by both In to this a bands were detected after These RNA not have translation of the by an scanning process for the no RNA was detected that but not with the that no has a 5′ end located downstream of the open reading that promote a cap-dependent translation of the and the of the bands were not after the from log to SIRES an in the expression of the in (see Fig. this be to of the We that the cells can an translation of the during by internal initiation of translation (see also the in the to Fig. the shown in and 4 that translation of the two that are by SIRES is Thus, the from log to when the translation of the first translation of the We after cells exit the log phase, SIRES the initiation of the translation from an internal site in the or no internal initiation can be detected in growing cells. experiments failed to IRESes in vegetatively growing yeast (see The of of IRESes in dividing with that the capacity to recognize an IRES is found in starved the that putative IRESes are not in optimally growing yeast cells but rather in or cells. from several that IRESes are when the initiation is The most are the observations that the 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 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 as a of the of the cap-dependent when yeast encounter the translation by more than 2 of (29Fuge E.K. Braun E.L. Werner-Washburne M. J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (135) Google Scholar, F. FEBS Lett. 1996; PubMed Scopus Google Scholar). We that a capacity to recognize putative natural has in yeast to a to the of the cap-dependent translation and that SIRES is by this Thus, putative natural are likely to the translation of encoding proteins that are for of the initiation SIRES can be utilized in the to that are capable of IRESes during starvation. also with the of natural It has been found that a yeast cell-free from logarithmically growing can recognize IRESes (14Altmann M. Blum S. Wilson T.M. Trachsel H. Gene. 1990; 91: 127-129Crossref PubMed Scopus (35) Google Scholar, N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar) or yeast sequences as IRESes (15Iizuka N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar). natural IRESes have not been identified in living yeast cells. for the of the in be to the is possible that the capacity to recognize IRESes also in log phase yeast and the in system this this capacity is repressed in dividing the with the or dividing cells or of both to this or more of is in of translation has been previously demonstrated in the of in Ref. A.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In a series of and A.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that induction of translation in to or is by short open reading in the leader of its The inhibit translation in cells by the of the scanning ribosomes the the scanning ribosomes the most and is to an translation of the A.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the of from that by SIRES. of translation from a that is by the the translation seems to be by of mRNA translation is that by SIRES is We that during when the 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, F. FEBS Lett. 1996; PubMed Scopus Google Scholar), are at two of mechanisms that mediate the translation of a small repertoire of mRNAs whose are for with starvation. We to O. S. and N. for reading the and A. Varshavsky and A. for and We are to all of for and