Dom34:Hbs1 Plays a General Role in Quality-Control Systems by Dissociation of a Stalled Ribosome at the 3′ End of Aberrant mRNA

Graduate School of Pharmaceutical Science, Tohoku University, Sendai, Japan.
Molecular cell (Impact Factor: 14.46). 04/2012; 46(4):518-29. DOI: 10.1016/j.molcel.2012.03.013
Source: PubMed

ABSTRACT Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.

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Available from: Toshifumi Inada, Apr 01, 2014
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    • "Nonsense-mediated mRNA decay (NMD) is one of three conserved eukaryotic surveillance pathways ensuring mRNA quality control in the cytoplasm [1] [2] [3] [4]. NMD is activated by mechanistic differences between normal and premature translation termination and utilizes three conserved factors (Upf1, Upf2, and Upf3) [5] [6] [7] to couple nonsense codon recognition to the release factors (eRF1 and eRF3), the ribosome, and the mRNA decapping complex [8] [9] [10] [11] [12] [13] [14]. "
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    ABSTRACT: Upf1, Upf2, and Upf3 are the principal regulators of Nonsense-mediated mRNA Decay (NMD), a cytoplasmic surveillance pathway that accelerates the degradation of mRNAs undergoing premature translation termination. These three proteins interact with each other, the ribosome, the translation termination machinery, and multiple mRNA decay factors, but the precise mechanism allowing the selective detection and degradation of nonsense-containing transcripts remains elusive. Here we have determined the crystal structure of the N-terminal mIF4G domain from Saccharomyces cerevisiae Upf2 and identified a highly conserved region in this domain that is essential for NMD and independent of Upf2's binding sites for Upf1 and Upf3. Mutations within this conserved region not only inactivate NMD, but also disrupt Upf2 binding to specific proteins, including Dbp6, a DEAD-box helicase. Although current models indicate that Upf2 functions principally as an activator of Upf1 and a bridge between Upf1 and Upf3, our data suggest that it may also serve as a platform for the association of additional factors that play roles in premature translation termination and NMD.
    Journal of Molecular Biology 09/2014; 426(22). DOI:10.1016/j.jmb.2014.09.015 · 4.33 Impact Factor
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    • "In our study, we split ribosomes using Pelota, Hbs1, and ABCE1, factors identified to recycle empty and stalled 80S ribosomes in earlier in vitro experiments (Pisareva et al., 2011). The physiologic targets of these factors are only now emerging and clearly include ribosomes at the end of truncated mRNAs (Guydosh and Green, 2014; Tsuboi et al., 2012). Because we employed a truncated mRNA in our experiments, the use of Pelota/Hbs1/ABCE1 is appropriate and physiologically relevant. "
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    ABSTRACT: Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain. Listerin alone can discriminate 60S- from 80S-nascent chain complexes to selectively ubiquitinate the former. Splitting factors can be bypassed by artificially removing the 40S subunit, suggesting that mere steric hindrance impedes Listerin recruitment. This was illustrated by a cryo-EM reconstruction of the 60S-Listerin complex that identifies a binding interface that clashes with the 40S ribosomal subunit. These results reveal the mechanistic logic of the core steps in a ribosome-associated quality control pathway.
    Molecular Cell 08/2014; 55(6). DOI:10.1016/j.molcel.2014.07.006 · 14.46 Impact Factor
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    • "The level of translated GFP- Rz was increased in ltn1D or ltn1Dnot4D mutant cells, but peptidyl-tRNA was barely detectable, even in the mutants (Fig. 1D), suggesting that peptidyltRNA on stalled 80S ribosomes might not be a substrate for the ligase, as previously proposed (Bengtson & Joazeiro 2010; Brandman et al. 2012). Dom34: Hbs1 is known to be required for the dissociation of ribosomes that are stalled at the 3′ end of GFP-Rz mRNA and for peptide release from tRNA in vivo (Tsuboi et al. 2012). To examine whether peptidyltRNA was stabilized in ltn1Ddom34D mutant cells, polysome analysis followed by NuPAGE and Western blotting with an anti-GFP antibody was carried out. "
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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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