Ebs1p, a Negative Regulator of Gene Expression Controlled by the Upf Proteins in the Yeast Saccharomyces cerevisiae

Abstract

Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-⌬ mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD.All eukaryotes have a nonsense-mediated mRNA decay (NMD) pathway that monitors the fidelity of gene expression at the level of RNA surveillance (6). One purpose served by NMD is to identify and degrade transcripts containing errors that cause premature termination of translation, which limits the accumulation of potentially deleterious truncated proteins (40). The UPF genes, which were first identified in Saccharomyces cerevisiae (8,26,27), are represented by orthologs in widely divergent evolutionary branches of the eukaryotes (6) but are absent in prokaryotes and archaea. In addition to RNA surveillance, the Upf proteins control the abundance of hundreds of transcripts in S. cerevisiae (18,28).The mechanism of nonsense transcript decay is intimately related to translation and decapping (31, 49). The Upf proteins and the translation termination factors eRF1 and eRF3 are assembled in a surveillance complex that is recruited to nonsense transcripts to catalyze termination at a premature stop codon, followed by decapping and decay (for a recent summary, see reference 7). Upf1p interacts with the decapping enzyme Dcp2p to promote Xrn1p-mediated decay from the 5Ј end (17, 36) and with Ski7p to promote exosome-mediated decay from the 3Ј end (32, 45). Although the broad outlines of the circuitry leading to NMD are known, the manner in which the surveillance complex affects the interplay between mRNA decay and translation is still unclear.To better understand the relationship between NMD and translation, we devised a selection method to uncover rare hypomorphic mutations in genes coding for either translation factors or additional Upf proteins. The possi...

Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance

“…As described earlier, ScEbs1 but not ScEst1 has been shown to render yeast cells rapamycin sensitive, possibly through a role in the NMD pathway (14,24). We therefore analyzed the rapamycin sensitivity of our KlEst1 mutants.…”

“…In S. cerevisiae, an EST1 homologue named EBS1 (Est1-like Bcy1 Suppressor) was shown to play a modest role in telomere maintenance but also to regulate translation and non-sense-mediated decay (NMD) (14,24,54). Specifically, the ebs1⌬ mutant manifests higher levels of NMD-targeted RNAs and greater resistance to rapamycin, which inhibits the TOR signaling pathway that regulates cell growth and translation.…”

Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry

“…we asked whether Ebs1p, a translation repressor that is down-regulated by the NMD pathway (Ford et al 2006), affects translation of the spliced GpII-CUP1 transcripts. Again, neither the ebs1 nor the ebs1 upf1 mutations suppressed the translation defect of the spliced GpII-CUP1 RNA (Fig.…”

Identification of <i>ebs1</i>, <i>lsm6</i> and <i>nup159</i> as suppressors of <i>spt</i>10 effects at <i>ADH</i>2 in <i>Saccharomyces cerevisiae</i> suggests post-transcriptional defects affect mRNA synthesis

“…We also showed that the lsm6, nup159 and ebs1 alleles did not alter the abundance of CCR4-NOT complex components, indicating that their effects were not indirectly through lowering CCR4 and CAF1 abundance in the cell. In addition, the role of EBS1 in negatively regulating translation [30] cannot directly explain how it would function as an activator of ADH2. spt10 results in a low level of ADH2 expression under glucose growth conditions by an unknown mechanism that could involve affecting ADH2 chromatin structure [2,35,36].…”

“…Its involvement in nonsense mediated decay may be only one means that its is involved in mRNA expression, as it has also been demonstrated that EBS1 binds eIF4E, the mRNA cap binding protein required for translational initiation. In fact, EBS1 is a negative regulator of translation [30]. EBS1 also binds DCP1 [31], a component of the mRNA decapping complex [20].…”