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: 4.87). 11/2007; 177(2):773-84. DOI: 10.1534/genetics.107.073205
Source: PubMed

ABSTRACT 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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    • "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 · 2.86 Impact Factor
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    • "a reporter, even though the protein A constituent of the TAP tag has been utilized as a reporter in analysis of non-stop mRNA decay, with quantification of protein A via Western blotting (Wilson et al., 2007). We have therefore verified that a TAP tag under the control of COX17 promotor and the ADH1 3 UTR was expressed at an easily detected level. "
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    ABSTRACT: Post-transcriptional regulation via the 3' untranslated region (3' UTR) of mRNA is an important factor in governing eukaryotic gene expression. Achieving detailed understanding of these processes requires highly quantitative systems in which comparative studies can be performed. To this end, we have developed a plasmid reporter system for Saccharomyces cerevisiae, in which the 3' UTR can be easily replaced and modified. Accurate quantification of the tandem affinity purification tag (TAP)-reporter protein and of TAP-mRNA is achieved by immuno-QPCR and by RT-QPCR, respectively. We have used our reporter system to evaluate the consequences on gene expression from varying the 3' UTR, a problem often encountered during C-terminal tagging of proteins. It was clear that the choice of 3' UTR was a strong determinant of the reporter expression, in a manner dependent on the growth conditions used. Mutations affecting either decapping (lsm1Delta) or deadenylation (pop2Delta) were also found to affect reporter gene expression in a highly 3' UTR-dependent manner. Our results using this set-up clearly indicate that the common strategy used for C-terminal tagging, with concomitant replacement of the native 3' UTR, will very likely provide incorrect conclusions on gene expression.
    Yeast 07/2009; 26(7):407-13. DOI:10.1002/yea.1675 · 1.74 Impact Factor
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    • "Strains ski7Δ, upf1Δ, and ski7Δupf1Δ yeast are isogenic to wild-type BY4741 (Open Biosystems). The ski7Δupf1Δ mutant was constructed by crossing ski7Δ::HYG (Wilson et al, 2007) to upf1Δ::NEO and sporulating. "
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    ABSTRACT: Eukaryotic mRNAs harboring premature translation termination codons are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. The mechanism for discriminating between mRNAs that terminate translation prematurely and those subject to termination at natural stop codons remains unclear. Studies in multiple organisms indicate that proximity of the termination codon to the 3' poly(A) tail and the poly(A) RNA-binding protein, PAB1, constitute the critical determinant in NMD substrate recognition. We demonstrate that mRNA in yeast lacking a poly(A) tail can be destabilized by introduction of a premature termination codon and, importantly, that this mRNA is a substrate of the NMD machinery. We further show that, in cells lacking Pab1p, mRNA substrate recognition and destabilization by NMD are intact. These results establish that neither the poly(A) tail nor PAB1 is required in yeast for discrimination of nonsense-codon-containing mRNA from normal by NMD.
    Molecular Cell 02/2008; 29(1):134-40. DOI:10.1016/j.molcel.2007.10.031 · 14.46 Impact Factor
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