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• W2019025253 abstract "A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae. A series of 5′-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability. The 5′-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene. The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA. Investigations of various derivatives of the 5′-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation. Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript. In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism. This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation. A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae. A series of 5′-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability. The 5′-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene. The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA. Investigations of various derivatives of the 5′-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation. Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript. In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism. This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation. Given that the translational machinery and the RNA degradation apparatus interact with the same mRNA molecules, it would come as no surprise if translation and mRNA decay were to mutually influence each other. It is obvious that mRNA degradation ultimately eliminates the template of translation. Yet the issue as to the role of translation in controlling mRNA decay is far from being resolved (1Sachs A.B. Cell. 1993; 74: 413-421Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 2Belasco J. Brawerman G. Control of Messenger RNA Stability. Academic Press, San Diego1993Google Scholar). This is not for the lack of indications that there could be a link between the two. (a) Rapid degradation of yeast MATα1 (3Caponigro G. Muhlrad D. Parker R. Mol. Cell. Biol. 1993; 13: 5141-5148Crossref PubMed Scopus (151) Google Scholar) and of mammalian early response genes (4Wellington C. Greenberg M.E. Belasco J.G. Mol. Cell. Biol. 1993; 13: 5034-5042Crossref PubMed Scopus (62) Google Scholar) is dependent on the translation of destabilizing elements within the respective coding regions. (b) Degradation of at least some yeast mRNAs is accelerated by nonsense codons introduced into the coding region (5Losson R. Lacroute F. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5134-5137Crossref PubMed Scopus (311) Google Scholar, 6Pelsey F. Lacroute F. Curr. Genet. 1984; 8: 277-282Crossref PubMed Scopus (35) Google Scholar, 7Peltz S.W. Brown A. Jacobson A. Genes & Dev. 1993; 7: 1737-1754Crossref PubMed Scopus (249) Google Scholar). Nonsense codons inserted into mammalian mRNAs seem to destabilize nuclear, rather than cytoplasmic, mRNA (8Cheng J. Maquat L. Mol. Cell. Biol. 1993; 13: 1892-1902Crossref PubMed Scopus (171) Google Scholar). In yeast, the so-called nonsense-mediated decay pathway is dependent on trans-acting factors (encoded by the UPF genes (9Leeds P. Wood J.M. Lee B.-S. Culbertson M.R. Mol. Cell. Biol. 1992; 12: 2165-2177Crossref PubMed Scopus (260) Google Scholar)), one of which (Upf1p) seems to be associated with ribosomes (10Peltz S.W. Trotta C. Feng H. Brown A. Donahue J. Welch E. Jacobson A. Brown A.J.P. Tuite M.F. McCarthy J.E.G. Protein Synthesis and Targeting in Yeast. Springer-Verlag, Berlin, Germany1993: 1-10Crossref Google Scholar). (c) Two means of inhibiting translation lead to stabilization of mRNA. These involve the inhibition of elongation using cycloheximide (1Sachs A.B. Cell. 1993; 74: 413-421Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 2Belasco J. Brawerman G. Control of Messenger RNA Stability. Academic Press, San Diego1993Google Scholar, 11Herrick D. Parker R. Jacobson A. Mol. Cell. Biol. 1990; 10: 2269-2284Crossref PubMed Scopus (321) Google Scholar, 12Peltz S.W. Donahue J.L. Jacobson A. Mol. Cell. Biol. 1992; 12: 5778-5784Crossref PubMed Scopus (68) Google Scholar) and the use of a mutation in tRNA nucleotidyl transferase (12Peltz S.W. Donahue J.L. Jacobson A. Mol. Cell. Biol. 1992; 12: 5778-5784Crossref PubMed Scopus (68) Google Scholar). Both of these experimental strategies impose a general, rather than an mRNA-specific, block on translation. (d) Finally, ribosomes have been found associated with certain ribonuclease activities (2Belasco J. Brawerman G. Control of Messenger RNA Stability. Academic Press, San Diego1993Google Scholar, 13Stevens A. J. Biol. Chem. 1980; 255: 3080-3085Abstract Full Text PDF PubMed Google Scholar). However, other data are not easily reconciled with the above observations. Most importantly, translational initiation can be inhibited by more than 90%, using stem-loops inserted into the 5′-untranslated region (UTR), 1The abbreviations used are:UTRuntranslated regionORFopen reading frameuORFupstream ORFCATchloramphenicol acetyltransferase. without stabilizing the mRNA (14Vega Laso M.R. Zhu D. Sagliocco F. Brown A.J.P. Tuite M.F. McCarthy J.E.G. J. Biol. Chem. 1993; 268: 6453-6462Abstract Full Text PDF PubMed Google Scholar, 15Sagliocco F.A. Zhu D. Vega Laso M.R. McCarthy J.E.G. Tuite M.F. Brown A.J.P. J. Biol. Chem. 1994; 269: 18630-18637Abstract Full Text PDF PubMed Google Scholar, 16Beelman C.A. Parker R. J. Biol. Chem. 1994; 269: 9687-9692Abstract Full Text PDF PubMed Google Scholar). This clearly constitutes a challenge to any model proposing tight coupling between translation and mRNA decay. untranslated region open reading frame upstream ORF chloramphenicol acetyltransferase. A required approach toward understanding the relationship between translation and mRNA degradation is to restrict engineered changes in the cell to minimal alterations in the sequence of a specific mRNA. This greatly reduces the risk of misleading or artifactual effects that might be associated with general manipulations of, for example, cellular translational capacity (see point c, above). The present paper approaches this question by making use of a further important property of the eukaryotic 5′-UTR. The presence of one or more additional start codons upstream of the main reading frame inhibits cap-dependent translation (17Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). This effect may be more pronounced in the yeast Saccharomyces cerevisiae, where the context of an AUG codon has a less significant modulating effect upon the efficiency of start-site selection than in higher eukaryotes (18Donahue T.F. Cigan M. Methods Enzymol. 1990; 193: 366-372Crossref Scopus (30) Google Scholar). An upstream AUG can be combined with a translational termination codon within the 5′-UTR, thus creating an upstream open reading frame (uORF). There are many examples of uORFs in the 5′-UTRs of vertebrate mRNAs (19Kozak M. J. Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1451) Google Scholar), and also a small number of them in S. cerevisiae (20Bossier P. Fernandes L. Rocha D. Rodrigues-Pousada C. J. Biol. Chem. 1993; 268: 23640-23645Abstract Full Text PDF PubMed Google Scholar, 21Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar). The GCN4 gene of yeast is of special interest in that it is subject to translational regulation mediated by four uORFs in its 5′-UTR (21Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar). The mechanism of induction of GCN4 involves phosphorylation of eIF-2α, which in turn inhibits the eIF-2B-catalyzed exchange of GDP/GTP on eIF-2. Regulation of the availability of eIF-2˙GTP in this way is thought to influence start-site selection within the leader. We show that the creation of a single short uORF in a synthetic 5′-UTR upstream of a reporter gene in yeast strongly inhibits translation. Moreover, given appropriate experimental design, a 5′-UTR that is otherwise free of uAUGs and of stable secondary structure can be converted to an uORF-containing leader by means of a single base change. This approach allows us to compare the translation and degradation of two mRNAs that differ minimally in terms of sequence but greatly in terms of the translational efficiency of a reporter gene (in this case the Tn9 cat gene). In comparison, strong inhibition of translation by secondary structure can only be achieved via the introduction or substitution of several nucleotides in the 5′-UTR. Further manipulations of the 5′-UTR were made in order to influence the pathway and efficiency of start-site selection on the main ORF, thus providing information about the type of mechanism responsible for the observed translation rates. We have investigated how the ribosomal loading of an mRNA is affected by the introduction of an uORF and how this relates to the decay rate. The results provide striking new evidence of a link between mRNA translation and decay. BWG1-7a (MATa, leu2-3, leu2-112, his4-519, ade1-100, ura3-52). Y262 (MATα, ura3-52, his4-539, rpb-1) (22Nonet M. Scafe C. Sexton J. Young R. Mol. Cell. Biol. 1987; 7: 1602-1611Crossref PubMed Scopus (268) Google Scholar). Yeast was cultured and transformed according to standard methods (23Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986: 117-122Google Scholar). The cloning of short upstream reading frames into the leader region of the cat mRNA was performed by introducing synthetic oligodeoxyribonucleotides into the YCpCATEX1 plasmid (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar) (see Table I). The oligonucleotide B1A, bearing an ORF of six codons, was inserted into the BamHI site at the 5′-end of the cat leader. A control construct was prepared using the synthetic B1O fragment, which differs from B1A in only one nucleotide (where B1A has AUG, B1O has AAG). The resulting leader does not contain an uORF. In order to increase the leader length upstream or downstream of the uORF, additional spacer fragments were cloned between the EcoRV and BamHI sites, respectively, creating USP10 and USP30. The leader length between the uORF and the cat coding region was increased by inserting a synthetic fragment between the XhoI and NdeI sites of B1A (DSP30). Further extension of the spacer region was achieved by inserting the 50-nucleotide fragment SP50 (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar) into the AflII site of DSP30, giving DSP80. OL136 was generated by destroying the termination codon of the uORF by means of digestion of YCpCATEX1-B1A with AflII, followed by treatment with mung bean nuclease and religation, yielding an extended uORF that overlaps with the cat gene, terminating 136 nucleotides downstream of the A of the cat start codon. uATG has three extra in-frame AUG codons upstream of the original B1A uORF start codon. B1ASS bears a secondary structure with a stability of −22 kcal/mol located inside the uORF, extending the uORF length from 6 to 21 codons. B1SS is a control for B1ASS, bearing the same secondary structure, but no uORF. B1X3 has been described elsewhere (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar) and was used as a control for B1AX3, which bears the uORF B1A and a secondary structure (X3) with a predicted stability of −17 kcal/mol, five nucleotides upstream of the cat start codon. The GCN4 leader, which bears four uORFs, was cloned upstream of the cat gene (after mutagenesis by PCR of the plasmid p180 (25Hinnebusch A.G. Mol. Cell. Biol. 1985; 5: 2349-2360Crossref PubMed Scopus (155) Google Scholar) in order to create a BamHI and an NdeI site at the 5′- and 3′-ends of the leader, respectively).Table I:Sequences of the 5′-UTRs Total yeast RNA was isolated by the hot phenol method (26Köhrer K. Dorndey H. Methods Enzymol. 1991; 194: 398-405Crossref PubMed Scopus (506) Google Scholar) from 150 ml of culture in YNB medium (0.67% bacto-yeast nitrogen base without amino acids, 2% dextrose) and analyzed using Northern blots after glyoxylation of 10-μg samples of total RNA, as described elsewhere (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar, 27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Primer extension analysis was performed as described previously (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar). mRNA half-life analysis (adapted from Ref. 28Parker R. Herrick D. Peltz S.W. Jacobson A. Methods Enzymol. 1991; 194: 415-423Crossref PubMed Scopus (91) Google Scholar) was performed using yeast transformants grown in 100 ml of YNB-lactate medium at 24°C for 18 h. Cells from these cultures were inoculated in the same volume of YNB-galactose and incubated at 24°C for 3 h to A600 = 0.8. Cells were then harvested by centrifugation and resuspended in the same volume of prewarmed (36°C) YNB-glucose medium. A 20-ml sample was collected at this time (time 0). The cell cultures were further incubated at 36°C in a shaking water bath, and samples were collected at various time points thereafter. Total RNA isolated from the different samples was used for Northern blots. The resulting labeled bands were excised from the blotting membrane and used for scintillation counting (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar). Fresh cultures of the yeast transformants were allowed to grow in YNB-galactose to A600 = 0.8-1.0. Cells from 10 ml of culture were used for analysis of CAT activity (adapted from Ref. 29Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5294) Google Scholar). After autoradiography of separated acetylated chloramphenicol forms, spots were counted in a scanning machine (linear analyzer, Chroma 2D, Berthold). Protein quantitation was performed by the BCA method (bicinchoninic acid protein assay (30Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18635) Google Scholar)). Yeast cells were grown in YNB-Gal medium to A600 = 0.3 (as described in Ref. 31Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (168) Google Scholar). 3-Aminotriazole (3-AT) was added to a final concentration of 10 m M, and incubation at 30°C was continued for another 6 h. Cells were harvested by centrifugation and washed with water, and the pellet was frozen and stored at −20°C until used for CAT assays and RNA analysis. In a procedure adapted from Sagliocco et al. (15Sagliocco F.A. Zhu D. Vega Laso M.R. McCarthy J.E.G. Tuite M.F. Brown A.J.P. J. Biol. Chem. 1994; 269: 18630-18637Abstract Full Text PDF PubMed Google Scholar), yeast cell extracts were prepared from cultures in 100 ml of YNB-Gal medium (A600 = 0.8) and loaded on a 12-ml diethyl pyrocarbonate 15-45% sucrose gradient. Total RNA was extracted from 600-μl fractions and resuspended in 10 μl of treated water. 5 μl of RNA suspension was glyoxylated and separated by electrophoresis in a 1.3% agarose gel. After blotting to a nylon membrane, Northern blot analysis was performed using 32P-labeled DNA fragments. Quantitation of the relative amounts of mRNA was performed as above. Short open reading frames (uORFs) were inserted into the leader region of the cat mRNA in the plasmid YCpCATEX1 (24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar) using a series of oligodeoxyribonucleotides (Fig. 1 and Table I). The presence of an uORF strongly inhibited translation of the cat gene, as seen, for example, by comparing the CAT levels of yeast cells bearing the constructs B1O and B1A (B1A supports 6.5% of the CAT activity supported by the control B1O; Fig. 2 A). These two constructs differ by only one nucleotide in the 5′-UTR.FIG. 2The relative CAT activities directed by the constructs illustrated in Fig. 1. Panel A shows the activities of all of the constructs relative to the activity of B10, which was normalized to the value of 1.00. Overall, the values demonstrate the strong inhibition of translation caused by the presence of an uORF in the 5′-UTR of the cat gene. The GCN4 construct bears the complete 5′-UTR from this yeast gene inserted upstream of the cat gene. Panel B compares the CAT activities of those constructs with an uORF in the 5′-UTR (except B1AX3). The data are grouped into two sections. The changes in encoded CAT activity relative to B10 in group 1 are most readily explained in terms of reductions in the amount of “leaky scanning” of preinitiation complexes past the uAUGs in the 5′-UTRs. At least part of the remaining cat translational activity is probably attributable to reinitiation of ribosomes subsequent to termination at the uORF stop codons. The changes in encoded CAT activity relative to B1A depicted in group 2 are more easily explained in terms of modulation of the efficiency of reinitiation at the cat start codon following termination on the uORF. The relative values schematically represented here (averages of measurements performed with at least three different sets of cell extracts) were as follows (±S.D. values): B1O, 1,00; B1A, 0.065 ± 0.010; USP10, 0.039 ± 0.007; USP30, 0.041 ± 0.009; DSP30, 0.067 ± 0.008; DSP80, 0.118 ± 0.025; uATG, 0.031 ± 0.006; OL136, 0.052 ± 0.013; B1SS, 0.020 ± 0.005; B1ASS, 0.007 ± 0.002; B1X3, 0.035 ± 0.008; B1AX3, 0.004 ± 0.001; GCN4, 0.011 ± 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Other constructs were designed with the intention of defining the possible processes responsible for translation of the cat gene (Fig. 2 B). The AUG codon of the uORF in B1A is located 13 nucleotides downstream of the 5′-end of the mRNA, which is a position thought not to allow efficient recognition of this AUG by the ribosome (17Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar, 18Donahue T.F. Cigan M. Methods Enzymol. 1990; 193: 366-372Crossref Scopus (30) Google Scholar). Increasing the leader length upstream of the B1A uORF (constructs USP10 and USP30; Fig. 1) resulted in further attenuation of cat expression. These results can be interpreted in terms of the scanning model of translation initiation (17Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). This would predict that translation of the cat gene in the B1A construct is due primarily to leaky scanning. It has been demonstrated that eukaryotic ribosomes need at least 14 nucleotides upstream of the AUG codon for efficient translation initiation to occur (32Sedman S.A. Gelembiuk G.W. Mertz J.E. J. Virol. 1990; 64: 453-457Crossref PubMed Google Scholar). Since USP10 and USP30 provide 23 and 43 nucleotides, respectively, upstream of the AUG of the uORF, the ribosomes can more efficiently recognize this AUG, initiating translation at the uORF and thus inhibiting cat translation more effectively (Fig. 2). By analogy, uATG, with its four in-frame AUGs (Fig. 1), should show decreased leaky scanning past the uORF and thus direct poorer translation of the cat reading frame. The observed reduction in cat translation relative to comparable constructs is consistent with leaky scanning providing many of the ribosomes for initiation on the cat ORF. Analysis of the above data alone does not rule out any contribution of reinitiation to the overall translation of cat. The described mutations in the leaders may not have eliminated leaky scanning completely. Further experimental results provided a stronger case for the occurrence of leaky scanning on the cat 5′-UTRs. Translating 80 S ribosomes are considered less sensitive to secondary structure than 40 S ribosomal subunits (33Liebhaber S.A. Cash F. Eshleman S.S. J. Mol. Biol. 1992; 226: 609-621Crossref PubMed Scopus (35) Google Scholar) and thus should be relatively insensitive to the presence of secondary structure within the uORF in B1ASS, providing that this structure is sufficiently distant from the uORF start codon to rule out any interference with initiation on the uORF. Thus if reinitiation was responsible for cat translation in B1A, translation of this gene in B1ASS would not be expected to be very different from that of B1A. However, the results show that translation of the cat gene is very strongly inhibited in B1ASS, indicating that the translation of the cat gene in B1A is probably due primarily to leaky scanning and not reinitiation (Fig. 2). Another potential effect of the inserted stem-loop structure should be considered. A stable secondary structure (−22 kcal/mol) positioned downstream of the uAUG, might even allow better recognition of this AUG (probably because it slows ribosome scanning in the vicinity of the start codon, compare Ref. 34Kozak M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8301-8305Crossref PubMed Scopus (405) Google Scholar), increasing levels of translation of the uORF. Thus in the case of a reinitiation mechanism, cat translation would have been expected to be at least as efficient in B1ASS as in B1A. Interestingly, the levels of cat expression in cells bearing B1ASS are more similar to those observed with B1SS, which contains the same secondary structure, but no uORF in the leader. Despite the observations discussed above, other evidence seems to indicate that reinitiation also occurs in B1A, albeit to a limited extent, and that its efficiency can be increased (Fig. 2 B). In constructs DSP30 and DSP80, the distance between the uORF and the cat gene was increased by 30 and 80 nucleotides, respectively. As argued by others (31Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (168) Google Scholar, 35Kozak M. Mol. Cell. Biol. 1987; 7: 3438-3445Crossref PubMed Scopus (406) Google Scholar), increasing the length between two ORFs may increase the probability that scanning ribosomes reinitiate translation at the downstream AUG. In this case, DSP30 and DSP80 should support higher levels of cat translation than B1A. This is indeed the result observed, although the difference between B1A and DSP30 is small (Fig. 2). The construct in which the uORF (+1 relative to the cat reading frame) overlaps the cat coding region (OL136) would be expected to support levels of cat expression not significantly different from that of B1A if leaky scanning was the dominant mechanism. However, if reinitiation was responsible for cat translation in B1A, the latter should decrease dramatically in OL136 (relative to B1A), since the termination codon of the uORF is inside the cat gene, 134 nucleotides downstream of the AUG. The translation of cat in OL136 was found to be reduced relative to B1A, although only by about 20% (Fig. 2). However, correction of the translation values for differences in mRNA abundance (see Fig. 5) would yield a greater inhibitory effect than this (approximately 35%). The B1AX3 construct (Fig. 1) does not allow differentiation between leaky scanning and reinitiation, but the results obtained with it indicate that the effects of the elements introduced into the leader act in series, as would be expected of a cap-dependent screening mechanism. In this construct, the presence of a secondary structure of predicted stability of −17 kcal/mol five nucleotides upstream of the cat initiation codon strongly inhibits translation (Fig. 2; compare Refs. 14Vega Laso M.R. Zhu D. Sagliocco F. Brown A.J.P. Tuite M.F. McCarthy J.E.G. J. Biol. Chem. 1993; 268: 6453-6462Abstract Full Text PDF PubMed Google Scholar and 24Oliveira C.C. van den Heuvel J. McCarthy J.E.G. Mol. Microbiol. 1993; 9: 521-532Crossref PubMed Scopus (63) Google Scholar). This degree of inhibition is approximately equivalent to the inhibitory effect of the uORFs present in most of the constructs. Fig. 2B summarizes the results obtained from the various CAT assays and indicates the simplest theoretical explanations of the respective effects observed. Given that the differences between the various CAT values are in some cases quite small, we do not wish to overinterpret the individual results described above, but would rather focus attention on the sum of these data. Overall, this indicates that translation of cat in B1A is largely due to leaky scanning. The remaining translation of the cat gene in B1ASS may be at least partially attributable to reinitiation, since the secondary structure is expected to strongly inhibit leaky scanning. The rate of reinitiation can apparently be increased by lengthening the spacer between the uORF and the cat start codon. For comparative purposes, we investigated the influence of the GCN4 leader on cat translation. It was found to direct a very low level of cat expression. Indeed, after correction for relative mRNA abundance (see Fig. 5 B), the translation rate of the GCN4 construct was lower than any of the other constructs tested. The long leader of this gene, containing four uORFs, evidently inhibits translation of the main ORF very effectively. Since the uORFs in the GCN4 leader are known to mediate up-regulation of this gene in response to amino acid starvation, we investigated how the single uORF leaders used in the present work respond under the same conditions. This situation can be simulated in the cell by adding 3-aminotriazole to the medium (31Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (168) Google Scholar). The GCN4 leader directed a 2.5-fold increase of cat expression upon addition of 3-aminotriazole. This result indicates that the starvation-induced reinitiation in the GCN4 leader continues to function when the cat gene is substituted for the main GCN4 ORF. In contrast, translation of the cat constructs bearing single uORFs was not influenced by the addition of 3-aminotriazole (data not shown). Clearly, the single uORF leaders do not fulfill the structural requirements of a system intended to respond to changes in eIF-2˙GTP availability. It could be argued that the uORF in B1A is poorly translated and that leaky scanning is the sole mechanism responsible for cat translation. This would mean that the complete leader as such would allow very restricted initiation (at whatever AUG codon). In order to investigate this more directly, we performed polysomal gradient analysis on cell extracts from cells carrying the constructs B1O, B1A, and OL136 (Figs. 3 and 4). The results demonstrate that the mRNA B1O is mainly associated with polysomes, corroborating the data from the CAT assays showing that this mRNA is efficiently translated (Fig. 3 A). In contrast, the B1A mRNA, which directs only low rates of CAT synthesis, was associated primarily with monosomes and disomes, showing only poor representation in the larger polysomal fractions. The data are compared directly with those of B1O in Fig. 4A. Despite the relatively low steady-state amount of B1A mRNA in the cell (see Fig. 5 B), it is evident that the cat Northern blot signal of this construct in the monosome/disome region is comparable with that of B1O. This result is consistent with relatively efficient initiation on the uORF of B1A, but not on the main cat ORF. Finally, the OL136 mRNA was found associated with larger polysomes than B1A (Figs. 3 B and 4 B). Again, this mRNA is relatively poorly represented in the cell (Fig. 5 B), yet the Northern blot signals (Fig. 4 B) in the monosomal and early polysomal fractions of the gradient are relatively strong. Thus the uORF in this construct was probably also translated considerably more efficiently than the cat coding region, but being considerably" @default.
• W2019025253 created "2016-06-24" @default.
• W2019025253 creator A5087805946 @default.
• W2019025253 creator A5091006816 @default.
• W2019025253 date "1995-04-01" @default.
• W2019025253 modified "2023-09-30" @default.
• W2019025253 title "The Relationship between Eukaryotic Translation and mRNA Stability" @default.
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• W2019025253 doi "https://doi.org/10.1074/jbc.270.15.8936" @default.
• W2019025253 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7721802" @default.
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