Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A
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PROVIDER: PRJNA565947 | ENA |
REPOSITORIES: ENA
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Project description:The Ccr4-Not complex is a conserved multi protein complex with diverse roles in the mRNA life cycle. Recently we determined that the Not1 and Not4 subunits of Ccr4-Not inversely regulate mRNA solubility and thereby impact dynamics of co-translation events. One mRNA whose solubility is most extremely inversely regulated by Not1 and Not4 is MMF1 encoding a mitochondrial protein that is translated with apparent ribosome pausing. In this work we uncover a mechanism that limits MMF1 overexpression in fermenting yeast and does not require MMF1 5’ or 3’ untranslated sequences but is mediated solely by the MMF1 coding sequence. This mechanism depends upon co-translational targeting of the MMF1 mRNA to the mitochondria, promoted by its mitochondrial targeting sequence, the Egd1 subunit of the NAC complex, the Om14 outer membrane protein and components of the mitochondrial import machinery as well as by Not4. Thereby, levels and translation of the MMF1 mRNA are limited by a mechanism that we have named Mito-ENCay involving ubiquitination of Egd1 by Not4, the Caf130 subunit of the Ccr4-Not complex and RQC. Our results indicate that this mechanism is used for highly expressed mRNAs that depend upon co-translational interactions for appropriate folding, such as ribosomal protein mRNAs in fermenting yeast.
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Project description:The conserved Ccr4-Not complex regulates all aspects of the RNA polymerase II (Pol II) gene expression pathway, while it also controls proteasome assembly and function. The Not4 RING domain ubiquitin ligase is a core subunit that also contains an RNA recognition motif (RRM) and C3H1 domain (collectively referred to as the RRM-C) whose function is unknown. Herein, we demonstrate that the Not4 RRM-C contributes to both Not4-dependent regulation of the proteasome and Pol II. Disruption of both Not4 ligase activity and the RRM-C domain simultaneously replicates the proteasome-dependent deubiquitylation and catalytic activity misregulation found in Not4-deficient cells. Transcriptome analysis reveals that the Not4 RRM-C affects a subset of Pol II-dependent genes involved in specific biological functions, including transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. At these genes, the Not4 RRM-C negatively regulates Pol II binding. While Not4 RRM-C disruption modestly increases between Ccr4-Not and Pol II, a Not4 ligase mutant decreases Ccr4-Not association with Pol II, thus implicating Not4-dependent ubiquitylation in mediating Ccr4-Not interaction with Pol II. Collectively, our results suggests the cellular RNA environment may integrate control of global proteostasis and Pol II transcription through the Not4 ubiquitin ligase.
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Project description:The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a substantial function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show accumulation at stop codons and in the 3’-UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of novel stalling sequences and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its great cellular abundance and essential nature.
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Project description:Not4 and Not5 are crucial components of the Ccr4-Not complex with pivotal functions in mRNA metabolism. Both associate with ribosomes but mechanistic insights on their function remain elusive. Here we determine that Not5 and Not4 synchronously impact translation initiation and Not5 alone alters translation elongation. Deletion of Not5 causes elongation defects in a codon-dependent fashion, increasing and decreasing the ribosome dwelling occupancy at minor and major codons respectively. This enhanced difference in codon translation velocities alters translation globally and enables normally kinetically unfavorable processes such as nascent chain deubiquitination to take place. In turn this leads to abortive translation and favors protein aggregation. These findings highlight the global impact of Not4 and Not5 in controlling the programmed local speed of mRNA translation.
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2008-06-13 | E-GEOD-5290 | biostudies-arrayexpress
Project description:eIF5A is an essential translation elongation factor present in all eukaryotes, and the only known protein to follow a post-translational modification called hypusination. Here, we performed a wide analysis of ribosome dynamics in S. cerevisiae eIF5A depleted cells using 5Pseq (Pelechano et al. 2015 PMID 26046441). This method allows the study of ribosome dynamics, by sequencing 5’ phosphorylated mRNA co-translational degradation intermediates. Since eIF5A is an essential protein in yeast, we used two eIF5A temperature-sensitive strains containing a single Pro83 to Ser mutation (tif51A-1) and double Cys39 to Tyr and Gly118 to Asp mutations (tif51A-3) in the highly expressed gene TIF51A (HYP2) that encodes eIF5A protein (Li et al. 2011 PMID: 24923804).
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Project description:Prior to type 2 diabetes onset, β cells adapt to insulin resistance through compensation—a process that maintains insulin secretion and glucose homeostasis. Our lab has previously shown that β cell compensation requires the activity of deoxyhypusine synthase (DHPS), which post-translationally catalyzes the formation of the amino acid hypusine at Lys50 of eukaryotic initiation factor eIF5A. Although hypusinated eIF5A is required for β cell compensation, it is unclear if unhypusinated eIF5A limits this compensatory response. To identify the role of unhypusinated eIF5A, we used the following animal and cell-based models: transgenic zebrafish and inducible, β cell-specific knockout mice fed a high fat diet. Zebrafish embryos injected with morpholinos to reduce global DHPS (and accumulate unhypusinated eIF5A) showed stunted exocrine pancreas growth at 3 days post-fertilization. Although, those injected with anti-eIF5A morpholinos (to deplete all eIF5A) showed normal pancreas growth. Although a unique function of unhypusinated eIF5A has not yet been documented, these findings suggest that the presence of unhypusinated eIF5A may be the major driver of altered pancreas phenotypes. Similarly, following 4 weeks of high fat diet feeding and obesity, mice lacking total eIF5A in β cells had improved glucose tolerance compared to mice lacking DHPS in β cells, despite similar weight gain and insulin sensitivity. Taken together, our data provide evidence that DHPS deficiency and obesity conditions impair β cell function, inpart, from the accumulation of the unhypusinated form of eIF5A. Our studies reveal a mechanism in which β cells respond to obesity by regulating mRNA translation through the balance between hypusinated and unhypusinated forms of eIF5A.
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