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A single amino acid substitution in yeast eIF-5A results in mRNA stabilization

1998; Springer Nature; Volume: 17; Issue: 10 Linguagem: Inglês

10.1093/emboj/17.10.2914

1460-2075

Dorit Zuk,

RNA and protein synthesis mechanisms

Article15 May 1998free access A single amino acid substitution in yeast eIF-5A results in mRNA stabilization Dorit Zuk Dorit Zuk Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655-0122 USA Search for more papers by this author Allan Jacobson Corresponding Author Allan Jacobson Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655-0122 USA Search for more papers by this author Dorit Zuk Dorit Zuk Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655-0122 USA Search for more papers by this author Allan Jacobson Corresponding Author Allan Jacobson Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655-0122 USA Search for more papers by this author Author Information Dorit Zuk1 and Allan Jacobson 1 1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655-0122 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2914-2925https://doi.org/10.1093/emboj/17.10.2914 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Most factors known to function in mRNA turnover are not essential for cell viability. To identify essential factors, ∼4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA. At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs. Of these, the mutant ts1159 had the most extensive phenotype. Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159. The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted α-helical segment of the protein. ts1159 also exhibited an ∼30% decrease in protein synthesis at the restrictive temperature. Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159. Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature. These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping. Introduction Differences in mRNA turnover rates can have significant effects on the expression of specific genes and provide a cell with flexibility in bringing about rapid changes in protein levels. The structures and mechanisms involved in the determination of individual mRNA decay rates have begun to be elucidated, particularly in yeast (Caponigro and Parker, 1996; Jacobson and Peltz, 1996). It is now well established that mRNA decay is a precise process dependent on a variety of specific cis-acting sequences and trans-acting factors. In addition, there is extensive evidence for an important role for translation in mRNA decay, including experiments demonstrating that: (i) inhibition of translational elongation can reduce mRNA decay rates (Peltz et al., 1992; Beelman and Parker, 1994); (ii) instability elements can be localized to mRNA coding regions (Parker and Jacobson, 1990; Caponigro et al., 1993; Hennigan and Jacobson, 1996; Jacobson and Peltz, 1996); (iii) the activity of some instability elements depends on ribosome translocation up to, or through the element (Parker and Jacobson, 1990; Peltz et al., 1993a; Hennigan and Jacobson, 1996); (iv) some 3′-untranslated region (UTR) instability elements can influence mRNA translational activity (Kruys et al., 1989; Marinx et al., 1993); (v) factors involved in decay and the decay process are polysome-associated (Peltz et al., 1993b; Caruccio and Ross, 1994; Atkin et al., 1995, 1997; Zhang et al., 1997); (vi) premature translational termination can enhance mRNA decay rates (Peltz et al., 1993a,b); (vii) factors essential for the rapid decay of nonsense-containing mRNAs also regulate translational suppression (Leeds et al., 1992; Cui et al., 1996; Weng et al., 1996); and (viii) metabolism of the poly(A) tail is a rate-limiting step in the decay of several mRNAs yet this structure and its binding protein also have a role in translational initiation (Jacobson, 1996; Sachs et al., 1997). The general outlines for at least three mRNA decay pathways have been delineated in the yeast Saccharomyces cerevisiae. In two of these pathways, mRNA decay proceeds from decapping to 5′→3′ exonucleolytic digestion (Decker and Parker, 1993, 1994), whereas, in the third pathway, the predominant event is 3′→5′ exonucleolytic digestion (Muhlrad et al., 1995). The initiation of decapping is known to be promoted by shortening of the mRNA poly(A) tail or premature translational termination (Muhlrad and Parker, 1994; Muhlrad et al., 1994). A number of trans-acting factors with demonstrated or implied roles in these pathways have been identified and include: a 5′→3′ exonuclease (Xrn1p; Hsu and Stevens, 1993), the decapping enzyme (Dcp1p; Stevens, 1988; Beelman et al., 1996; La Grandeur et al., 1998), poly(A)-binding protein (Pab1p; Adam et al., 1986; Sachs et al., 1986), constituents of a poly(A) nuclease complex (Pan2p and Pan3p; Boeck et al., 1996; Brown et al., 1996), two proteins that regulate decapping (Mrt1p and Mrt3p; Hatfield et al., 1996) and a number of factors that are required specifically for the decay of nonsense-containing mRNAs (Upf1p, Nmd2p/Upf2p and Upf3p; Peltz et al., 1994; Cui et al., 1995; He and Jacobson, 1995; Lee and Culbertson, 1995; Lee et al., 1995; He et al., 1997). Only three of these mRNA decay factors (Pab1p, Mrt1p and Mrt3p) are encoded by essential genes (Adam et al., 1986; Sachs et al., 1986; Hatfield et al., 1996). In contrast, many genes involved in other complex cell processes, such as transcription or translation, are essential. In order to identify other essential factors involved in mRNA decay, we screened a yeast library of ∼4000 temperature-sensitive (ts) mutants for those that were incapable of degrading either wild-type or nonsense-containing mRNAs at the restrictive temperature. We have identified several mutants in which a shift to the non-permissive temperature promotes mRNA stabilization and report here on the ts strain displaying the most extensive impairment of mRNA decay. That strain, ts1159, was found to harbor a mutation in TIF51A, the gene encoding eIF-5A. Results Identification of temperature-sensitive strains that selectively accumulate the unstable CYH2 pre-mRNA The collection of yeast strains used in this study was generated by J.Abelson and colleagues in a study of ts mutants defective in pre-mRNA splicing. The yeast strain SS330 was treated with ethylmethylsulfonate (EMS), and ∼4000 mutants capable of growth at 24°C, but not at 37°C, were isolated (Vijayraghavan et al., 1989; J.Abelson, personal communication). Northern blots of RNA isolated from each mutant after a 2 h shift to the non-permissive temperature were then hybridized to an ACT1 probe. This led to the identification of a number of new splicing mutants that accumulated the ACT1 pre-mRNA at 37°C (Ruby and Abelson, 1991). The original collection of blots was rescreened to identify mutants in which mRNA was stabilized at 37°C. Earlier work had shown that endogenous substrates of the nonsense-mediated mRNA decay pathway include inefficiently spliced pre-mRNAs that enter the cytoplasm (such as the CYH2 pre-mRNA; He et al., 1993). Mutants incapable of nonsense-mediated mRNA decay, i.e. strains containing deletions of the UPF1, NMD2 or UPF3 genes, lead to the stabilization and selective accumulation of such RNAs (He et al., 1993; Peltz et al., 1994; He and Jacobson, 1995) and we expected that strains with ts lesions in this pathway would have related phenotypes. The blots were thus hybridized with probes for CYH2 and also for ACT1 transcripts (the latter to minimize the chance of isolating splicing mutants). We identified 96 mutants that accumulated the CYH2 pre-mRNA, but not the ACT1 pre-mRNA, at the restrictive temperature (data not shown). The RNA in the original collection of Northern blots had been prepared only from cells that had been shifted to the non-permissive temperature for 2 h. In order to directly compare the levels of the CYH2 transcripts in these cells before and after the change in temperature, we prepared RNA from aliquots of these strains grown at 24°C or incubated at 37°C for 45 min. Northern blots of these RNA preparations were probed with radiolabeled CYH2 DNA. Figure 1 shows an example of these blots including seven ts mutants and a splicing mutant (prp2) control. Three mutants, ts1159, ts1189 and ts1197, exhibited an increase in the level of the CYH2 pre-mRNA after the shift to the non-permissive temperature and were thus chosen for additional testing. Nine other strains (ts817, ts942, ts970, ts1100, ts1387, ts1581, ts3753, ts3766 and ts3771) were chosen from additional blots by the same criteria (data not shown). Figure 1.CYH2 pre-mRNA levels increase in some mutants grown at 37°C. RNA was prepared from cells of the indicated ts mutant strains grown at 24°C or shifted to 37°C as described in Materials and methods. Northern blots containing 15 μg of RNA per lane were hybridized to a radiolabeled CYH2 probe. pre-mRNA and mRNA depict the respective CYH2 transcripts. Download figure Download PowerPoint Identification of mutants with temperature-sensitive defects in mRNA decay Increases in CYH2 pre-mRNA abundance could be attributable to changes in the rates of decay or synthesis of that transcript. To distinguish between these possibilities for the selected mutants, and to characterize their mRNA decay defects further, we analyzed the kinetics of turnover of a number of mRNAs in these strains after a shift to the non-permissive temperature. The cells were grown at 24°C to mid-log phase and shifted to 37°C for 1 h. Transcription was then inhibited by the addition of thiolutin (Jimenez et al., 1973). RNA was isolated 0, 5, 10, 20 and 35 min after inhibition of transcription and analyzed by Northern blotting, using probes for five transcripts (Herrick et al., 1990). These represented different classes of transcripts found in the cell and included the CYH2 pre-mRNA and the CYH2, STE2, PAB1 and URA5 mRNAs. The CYH2 pre-mRNA, as noted above, is a substrate of the nonsense-mediated mRNA decay pathway, the STE2 and URA5 mRNAs have short half-lives (<7 min) and in this strain, the PAB1 and CYH2 mRNAs are moderately stable transcripts (Herrick et al., 1990). Seven of the mutant strains tested in this manner (ts970, ts1197, ts1387, ts1581, ts3753, ts3766 and ts3771) did not show a defect in the decay rates of the mRNAs tested. They were thus deemed not to be mRNA-turnover mutants and were not characterized further (data not shown). The remaining mutants (ts817, ts942, ts1100, ts1159 and ts1189) exhibited decreased decay rates of some or all of the transcripts tested when compared with the rates measured in the wild-type parental strain, SS330. Of the latter set of mutants, ts1159 exhibited the most substantial effects on mRNA decay, stabilizing some mRNAs up to 5-fold (Figure 2). Figure 2.mRNA half-lives are increased in ts1159 cells shifted to 37°C. Wild-type (SS330) and ts1159 cells were grown at 24°C, shifted to 37°C for 1 h, and then incubated with 4 μg/ml thiolutin (see Materials and methods). Northern blots were prepared from RNAs extracted from aliquots taken at the indicated time points thereafter (Time after TL) and hybridized to a series of radiolabeled probes (indicated to the left of the blots). The half-lives (t1/2) of each transcript were calculated and are listed to the right of each panel. Download figure Download PowerPoint Identification of the gene mutated in ts1159 The ts growth phenotypes of the five strains that exhibited mRNA decay defects were complemented by a yeast genomic DNA library (cloned in the centromere vector YCp50; Rose et al., 1987) and plasmids capable of inducing temperature-independent growth in each strain were identified. Transformation of ts1159 cells with the YCp50 library yielded three clones (p1159-1 to p1159-3) capable of growth at the restrictive temperature. Cells from the original ts1159 strain, or from that strain transformed with a complementing plasmid, were patched onto rich, synthetic complete (SC) or SC-ura plates, grown for 3 days at 24°C, and then replica-plated to two plates, one of which was incubated at 24°C and the other at 37°C, each for 3 days. Expression of the relevant YCp50 clones was shown to restore growth at 37°C to the mutant cells (Figure 3A). Moreover, growth at the higher temperature was shown in two ways to be plasmid dependent: (i) by isolation of the respective plasmids, retransformation into fresh mutant cells and verification of the complementation phenotype; and (ii) by growing complemented cells to saturation in non-selective media and then showing that all clones that had lost the plasmid also lost the ability to grow at 37°C (data not shown). Figure 3.A YCp50 library clone complements the ts growth and mRNA decay phenotypes of ts1159 at 37°C. (A) ts1159 cells were patched onto SC plates and grown at 24°C for 3 days. These were replica-plated to two SC plates, and grown at 24 and 37°C, respectively (left panels). A similar experiment (on SC-ura plates) was done with ts1159 cells transformed with the YCp50 plasmid 1159-1 (right panels). (B) ts1159 cells, with and without the 1159-1 plasmid, were grown at 24°C or shifted to 37°C for 1 h. RNA was isolated from the respective cells and analyzed by Northern blotting, using a radiolabeled STE2 probe. The ethidium bromide-stained rRNAs, indicating the amount of RNA in each sample, are shown under each lane. Download figure Download PowerPoint In order to verify that the ts growth phenotype was linked to the mRNA decay defect, ts1159 cells with and without a complementing plasmid were grown at 24°C and shifted to 37°C for 1 h. RNA prepared from these cells was analyzed by Northern blotting, using an STE2 fragment as a hybridization probe. There is a 3.3-fold increase in the level of the STE2 mRNA in ts1159 cells after a shift to the non-permissive temperature (Figure 3B), but this increase is not evident in cells harboring the complementing 1159-1 YCp50 plasmid. This indicates that the same plasmid which complemented the ts growth phenotype also complemented the mRNA decay defect. Initial mapping of the three plasmids rescued from the complemented cells using the restriction enzymes EcoRI and SalI showed that they comprised two different plasmids (one represented twice, and the other once; data not shown). The vector–insert junctions of the two plasmids (1159-1 and 1159-3) were sequenced, compared with the available databases and found to contain overlapping fragments of chromosome V. The overlapping sequence included three open reading frames (ORFs) of unknown function (yEL033w, yEL035c and yEL038w), a serine tRNA gene, the TIF51A (HYP2) gene (which encodes the putative translation initiation factor eIF-5A; Kemper et al., 1976; Benne et al., 1978; Schnier et al., 1991), the ANP1 gene (which encodes an endoplasmic reticulum protein; Chapman and Munro, 1994) and the RAD23 gene (involved in DNA repair; Miller et al., 1982). The ORFs contained in the complementing plasmids were then subcloned separately into the centromere vector pRS316 (Sikorski and Hieter, 1989), along with a substantial length of 5′ and 3′ sequences to ensure inclusion of the regulatory sequences flanking each gene. Each ORF construct was transformed into ts1159 cells and tested for complementation of the growth defect at 37°C. Only one plasmid (pDZ4), expressing the TIF51A ORF, was capable of complementing the growth of the mutant strain at the non-permissive temperature. ts1159 cells transformed either with the pRS316 vector alone or with the complementing TIF51A plasmid were patched onto SC-ura plates, grown for 3 days at 24°C, and then replica-plated to two plates, one of which was incubated at 24°C and the other kept at 37°C, each for 3 days. It is evident from the data shown in Figure 4A that the construct expressing TIF51A restored the growth at 37°C, while the vector alone could not complement the growth defect. Figure 4.A plasmid expressing TIF51A complements the ts growth and mRNA decay phenotypes of ts1159. Experiments similar to those described in Figure 3 were done with ts1159 cells transformed with pRS316 or with pRS316 containing TIF51A (pDZ4). (A) Growth at 24 and 37°C. (B) Northern blots of the cells grown at 24°C, and after a 1 h shift to 37°C, using a STE2 fragment as a hybridization probe. The ethidium bromide-stained rRNAs, indicating the amount of RNA in each sample, are shown under each lane. Download figure Download PowerPoint In order to ensure that the complementation of the ts growth phenotype and mRNA decay defect resulted from the same ORF on the original YCp50 clone, ts1159 cells transformed with the empty vector and with pDZ4 were grown at 24°C and shifted to 37°C for 1 h. RNA prepared from these cells was again analyzed by Northern blotting. As can be seen in Figure 4B, there is a 3-fold increase in the level of the STE2 mRNA in the ts1159 cells transformed with pRS316 after a shift to the non-permissive temperature (as was seen for the untransformed ts mutant in Figure 3B), but this increase does not occur in the cells harboring the plasmid expressing TIF51A. This indicates that both the growth and mRNA decay defects were complemented by expression of a single gene. Comparable experiments identified the genes complementing the other four mutants (Table I). SLA2 encodes a protein containing a talin-like domain, which previously was identified as being involved in membrane cytoskeletal assembly, endocytosis of α-factor and maintenance of the PMA1 ATPase (Holtzman et al., 1993; Raths et al., 1993; Na et al., 1995). GRC5 (also called QSR1 and QM1) encodes the 60S ribosomal subunit protein L9 (Dick et al., 1997; Nika et al., 1997), and THS1 encodes the yeast threonyl-tRNA synthetase (Pape and Tzagoloff, 1985). MRT4 (mRNA turnover; yKL009w) is a previously uncharacterized gene which encodes a protein similar to the P0/L10 family of 60S ribosomal proteins. Here we report on the initial characterization of the ts1159 mutant and the tif51A allele found in that strain. Further studies of the other four mutants will be reported elsewhere. Table 1. Complementation of ts mutants deficient in mRNA decay ts mutant Chromosome Complementing ORF ts817 XIV SLA2 ts 942 XII GRC5 ts1100 IX THS1 ts1159 V TIF51A ts1189 XI MRT4 The indicated ORFs plus enough flanking sequence to overlap with neighboring ORFs were cloned into the pRS316 vector and expressed in the relevant strain. Complementing = allowing growth at the non-permissive temperature. Isolation of the mutated tif51A allele from ts1159 To identify the specific mutation in the ts1159 tif51A allele (which we designate tif51A-1159), we rescued the mutation by gap repair. The pDZ4 construct contains the 473 nucleotide TIF51A ORF plus 193 nucleotide upstream and 105 nucleotide downstream sequences in the URA3-based vector, pRS316 (see Materials and methods). We generated two types of pDZ4 plasmids containing 74 or 209 nucleotide gaps at the 3′ end of the gene (with the latter encompassing the former). These plasmids were transformed into ts1159 cells, ura+ colonies were selected, and the repaired plasmids (which now included sequence from the chromosomal copy of tif51A-1159) were isolated. Re-transformation of the wild-type and repaired alleles into fresh ts1159 cells showed that the repaired tif51A-1159 allele [pDZ4(g-r)] could no longer complement the ts growth phenotype of the ts1159 mutant when compared with the complementation observed in cells transformed with the original TIF51A gene (Figure 5A). In order to test for complementation of the mRNA turnover phenotype, cells harboring the pDZ4 wild-type allele, the repaired plasmid or no plasmid were grown at 24°C, shifted to 37°C for 1 h and then tested for levels of STE2 mRNA by Northern blotting. The results in Figure 5B reiterate those shown in Figure 4B, i.e. the ∼3-fold increase in STE2 mRNA levels in the ts1159 cells at 37°C is abrogated by the expression of the wild-type TIF51A allele (pDZ4). However, this increase is not reversed by expression of the gap-repaired pDZ4(g-r) construct. Figure 5.The tif51A allele isolated from ts1159 cells is unable to complement the ts growth and mRNA decay phenotypes of this strain. (A) ts1159 cells transformed with the construct expressing the wild-type TIF51A allele (pDZ4; left panels) or with the large gap-repaired construct expressing the tif51A allele rescued from the ts mutant [pDZ4(g-r); right panels] were grown on SC-ura plates at 24°C for 3 days. These were replica-plated to two SC plates, and grown at 24 and 37°C, respectively. (B) ts1159 cells that were untransformed, harboring the pDZ4 construct, or harboring the gap-repaired construct [pDZ4(g-r)] were grown at 24°C or shifted to 37°C for 1 h. RNA was isolated and analyzed by Northern blotting, using a STE2 probe. The ethidium bromide-stained rRNAs, indicating the amount of RNA in each sample, are shown under each lane. (C) The sequence of the insert in the pDZ4 and pDZ4(g-r) constructs. The sequence of the eIF-5A protein is listed beneath the DNA sequence. The ATG translation start and the TAA translation stop sites are indicated by double underlines. The large gap created in the pDZ4 plasmid (which was then repaired in the ts1159 cells) is underlined. The T→C (S→P) mutation is indicated above (and below) the sequence and italicized. Download figure Download PowerPoint Sequencing of the gap-repaired plasmids revealed a single T→C change at position 445 of the TIF51A ORF (Figure 5C), which resulted in a serine to proline change at position 149 of the eIF-5A protein. The remaining chromosomal sequence in the repaired gap is the same as in the construct expressing the wild-type TIF51A allele, as is the sequence in the rest of the pDZ4 insert. Therefore, we conclude that the lack of complementation of both the growth and mRNA decay phenotypes in the ts1159 cells expressing the gap-repaired plasmid is due to this single amino acid change. The results of this experiment and of the complementation experiments shown in Figure 4 support the notion that both the growth and mRNA decay phenotypes are due to the same mutation in the TIF51A gene. Additional evidence for mutation of a single gene comes from the fact that tetrads resulting from sporulation of a cross between the ts1159 strain and the SS328 parent strain segregate 2:2 for ts growth (data not shown). Protein synthesis properties of the mutant strain Since the eIF-5A protein was isolated initially from a ribosomal fraction of rabbit reticulocytes, it was considered a translation initiation factor (Kemper et al., 1976; Benne et al., 1978). It was thus important to investigate whether the ts1159 strain was defective in protein synthesis. Cytoplasmic extracts of ts1159 and wild-type cells before and after a shift to the non-permissive temperature were fractionated on sucrose gradients and ribosome distribution was examined. The wild-type cells display a polysome peak of approximately eight ribosomes at both 24°C and after a 1 h shift to 37°C (Figure 6A and B, respectively), with a slight increase in the amount of 80S subunits after the shift to the restrictive temperature. The profiles in the ts1159 mutant at both temperatures look very similar to those seen in the wild-type extracts, except that polysomes now peak at approximately six ribosomes (Figure 6C and D). These experiments suggest that any temperature-induced translation defect in the ts1159 mutant is not extensive and, because of the absence of a substantial shift to smaller polysomes at 37°C, is not likely to have a significant effect on translation initiation. Figure 6.Polysome profiles are not altered in ts1159 cells after a shift to 37°C. Cytoplasmic extracts were prepared from wild-type (A and B) and ts1159 (C and D) cells grown at 24°C (A and C) or after a shift to 37°C for 1 h (B and D) and fractionated on sucrose gradients as described in Materials and methods. The OD trace of the gradients is shown, with the direction of sedimentation and the 80S, 60S and 40S peaks indicated by arrows. Download figure Download PowerPoint In order to study the protein synthesis properties of the ts1159 cells in a more quantitative manner, we measured the incorporation of 35S-labeled amino acids (methionine and cysteine) into cells after a shift to the non-permissive temperature. Duplicate aliquots of cells were taken at various time points after the shift, incubated with the labeled amino acids, and then precipitated and washed with trichloroacetic acid (TCA). The resulting precipitates were counted and compared with the amount of label incorporated at the time of the shift to 37°C (t = 0). The results of these experiments are shown in Figure 7. It is evident that protein synthesis in the wild-type cells increases after 60 min at the higher temperature (after a modest drop at 30 min, probably due to the heat shock of the shift to 37°C). The ts1159 (tif51A-1159) cells display a drop in translation of ∼30% after 60 min at the non-permissive temperature (the time at which decay rates were measured; Figure 2). Figure 7.Amino acid incorporation in ts1159 and wild-type cells. Incorporation of 35S-labeled amino acids was measured in SS330 wild-type (WT) and ts1159 cells as described in Materials and methods. The values are given as the percentage incorporation, with time zero taken as 100%, and are averages of duplicate samples. The error bars denote the standard deviations of three separate experiments. Filled bars, time zero after the shift to 37°C; hatched bars, 30 min after the shift; open bars, 60 min after the shift. At t = 0, the average c.p.m. for wild-type and mutant cells were 47 970 and 24 092, respectively. Download figure Download PowerPoint Effect of general inhibition of translation on the accumulation of mRNAs While the results of the polysome gradients and the amino acid incorporation assays suggest that any translation defects in ts1159 are modest (Figures 6 and 7), they nevertheless raise the question of whether the mRNA decay phenotype observed in this strain is an indirect consequence of impaired protein synthesis. Previous work has shown that complete inhibition of protein synthesis results in a general stabilization of all cellular mRNAs (Herrick et al., 1990; Peltz et al., 1992; Beelman and Parker, 1994). However, the effects on mRNA decay rates of a partial deficiency in translation have not been characterized previously. In order to investigate further the correlation between the extent of inhibition of protein synthesis and the resulting stabilization of specific mRNAs, we used the drug cycloheximide as a general inhibitor of translation. The SS330 parental strain was incubated with increasing concentrations of cycloheximide (ranging from 0.05–0.8 μg/ml) for 1 h, and then duplicate aliquots were allowed to incorporate 35S-labeled amino acids. RNA was prepared from the same cultures, and Northern blots of these RNAs were probed with the CYH2 and URA5 probes. The results are shown in Figure 8. It is evident that inhibition of protein synthesis by 30% results in a <2-fold stabilization of the three transcripts tested. The ts1159 cells, which have a similar defect in protein synthesis, exhibited a 2.4-fold increase in the half-life of the CYH2 pre-mRNA, a 5-fold increase in that of the URA5 mRNA and a 5.2-fold increase in the half-life of the CYH2 mRNA (Figure 2). A similar 4- to 5-fold increase in the abundance of the CYH2 and URA5 mRNAs was only observed in the cycloheximide experiments when the inhibition of protein synthesis reached 80% (Figure 8). These results, taken together with the virtually normal polysome profiles of the ts1159 cells at the non-permissive temperature, suggest that the mRNA decay defects displayed by ts1159 are at least partially a result of a defect in some metabolic process other than protein synthesis. Figure 8.Extent of mRNA stabilization in cycloheximide-treated cells. Top: SS330 wild-type cells were grown in SC-met and incubated with the indicated concentrations of cycloheximide for 1 h at 30°C. Incorporation of 35S-labeled amino acids was measured in the cells as described in Materials and methods. The values are depicted as the percentage incorporation (thick dashed line, left scale), with the c.p.m. obtained from the culture lacking cycloheximide taken as 100%. RNA was isolated from the same samples, run on Northern blots and hybridized to radiolabeled URA5 and CYH2 probes. The values are depicted as fold stabilization (thin lines, right scale), with the amount of RNA in the sample lacking cycloheximide defined as 1. Bottom: Northern blots from which the values in the top panel were derived. Download figure Download PowerPoint