A Genomic Screen in Yeast Reveals Novel Aspects of Nonstop mRNA Metabolism

Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030, USA.
Genetics (Impact Factor: 5.96). 11/2007; 177(2):773-84. DOI: 10.1534/genetics.107.073205
Source: PubMed


Nonstop mRNA decay, a specific mRNA surveillance pathway, rapidly degrades transcripts that lack in-frame stop codons. The cytoplasmic exosome, a complex of 3'-5' exoribonucleases involved in RNA degradation and processing events, degrades nonstop transcripts. To further understand how nonstop mRNAs are recognized and degraded, we performed a genomewide screen for nonessential genes that are required for nonstop mRNA decay. We identified 16 genes that affect the expression of two different nonstop reporters. Most of these genes affected the stability of a nonstop mRNA reporter. Additionally, three mutations that affected nonstop gene expression without stabilizing nonstop mRNA levels implicated the proteasome. This finding not only suggested that the proteasome may degrade proteins encoded by nonstop mRNAs, but also supported previous observations that rapid decay of nonstop mRNAs cannot fully explain the lack of the encoded proteins. Further, we show that the proteasome and Ski7p affected expression of nonstop reporter genes independently of each other. In addition, our results implicate inositol 1,3,4,5,6-pentakisphosphate as an inhibitor of nonstop mRNA decay.

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    • "The E3 ligase Ltn1 plays a crucial role in the degradation of arrest products produced by the translation of poly(A) sequences (Wilson et al., 2007; Bengtson and Joazeiro, 2010) and derived from mRNAs containing arrest-inducing sequences, including polybasic amino acid sequences and rare codons (Matsuda et al., 2014). Ltn1 may recognize peptidyl-tRNA on stalled ribosomes and ubiquitinate this complex for degradation by the proteasome. "
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    ABSTRACT: The CCR4-NOT complex is a highly conserved specific gene silencer that also serves more general post-transcriptional functions. Specific regulatory proteins including the miRNA-induced silencing complex and its associated proteins, bind to 3'-UTR elements of mRNA and recruit the CCR4-NOT complex thereby promoting poly(A) shortening and repressing translation and/or mRNA degradation. Recent studies have shown that the CCR4-NOT complex that is tethered to mRNA by such regulator(s) represses translation and facilitates mRNA decay independent of a poly(A) tail and its shortening. In addition to deadenylase activity, the CCR4-NOT complex also has an E3 ubiquitin ligase activity and is involved in a novel protein quality control system, i.e., co-translational proteasomal-degradation of aberrant proteins. In this review, we describe recent progress in elucidation of novel roles of the multi-functional complex CCR4-NOT in post-transcriptional regulation.
    Frontiers in Genetics 05/2014; 5:135. DOI:10.3389/fgene.2014.00135
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    • "Surprisingly, however, ubiquitination of ribosome-associated translationally arrested polypeptides were increased in the absence of Not4, an observation that is inconsistent with a role in cotranslational ubiquitination (Duttler et al., 2013). The importance of Not4 for proteasome functional integrity may explain this last observation since proteasome inhibition or mutation also leads to the accumulation of polyubiquitinated protein products arising due to translational arrest (Wilson et al., 2007; Dimitrova et al., 2009; Bengtson and Joazeiro, 2010). This supports a role for Not4 in cotranslational protein QC via clearance of aberrant proteins via the proteasome. "
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    ABSTRACT: Cotranslational quality control (QC) is the mechanism by which the cell checks the integrity of newly synthesized proteins and mRNAs. In the event of mistakes these molecules are degraded. The Ccr4-Not complex has been proposed to play a role in this process. It contains both deadenylation and ubiquitination activities, thus it may target both aberrant proteins and mRNAs. Deadenylation is the first step in mRNA degradation. In yeast it is performed by the Ccr4 subunit of the Ccr4-Not complex. Another complex subunit, namely Not4, is a RING E3 ligase and it provides the ubiquitination activity of the complex. It was found associated with translating ribosomes. Thus, it has been suggested that Not4 is involved in ribosome-associated ubiquitination and degradation of aberrant peptides. However, several other E3 ligases have been associated with peptide ubiquitination on the ribosome and the relevance of Not4 in this process remains unclear. In this review we summarize the recent data and suggest a role for Not4 in cotranslational protein QC.
    Frontiers in Genetics 05/2014; 5:141. DOI:10.3389/fgene.2014.00141
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    • "In addition, Not4 associates with polyribosomes, as revealed by polysome fractionation (Dimitrova et al. 2009; Panasenko & Collart 2012), and is involved in protein degradation of the translation arrest products produced by poly-lysine sequences, but not those of nonstop proteins (Dimitrova et al. 2009). Ltn1 is also involved in the co-translational protein degradation of arrest products produced by the translation of poly(A) sequences (Wilson et al. 2007; Bengtson & Joazeiro 2010). The mechanisms by which ubiquitin ligases recognize aberrant products produced from aberrant mRNAs have been investigated. "
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    ABSTRACT: Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.
    Genes to Cells 11/2013; 19(1). DOI:10.1111/gtc.12106 · 2.81 Impact Factor
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