Reserach / 研究の概要

  • Quality Control of Aberrant mRNAs
  • Translation Control by Nascent peptide
  • Co-translational Degradation of mRNA and Protein
  • Mechanism of DNA replication, repair, and recombination
  • Chromatin dynamics and DNA transactions

Graduate School of Pharmaceutical Sciences,
Tohoku University,
Sendai 980-8578, Japan
TEL:022-795-6874; FAX:022-795-6873
e-mail:tinada@m.tohoku.ac.jp

Gene expression is highly accurate due to quality-control systems that prevent the expression of potentially harmful protein products. We previously reported that translation of the poly(A) tail plays a crucial role in repressing the production of aberrant proteins from nonstop mRNAs by both translation repression and proteasome-dependent nascent protein destabilization in yeast (Inada et al., 2005; Ito-Harashima et al., 2007). This clearly indicates that translation arrest and protein degradation, in addition to mRNA degradation, are crucial for repressing the expression of nonstop mRNAs. We also recently reported that translation arrest caused by the presence of consecutive basic amino acids in a nascent protein induces Not4p-dependent co-translational protein degradation by the proteasome (Dimitrova et al., 2009). In addition, the receptor for activated C kinase (RACK1) participates in nascent peptide-dependent translation arrest, and that the binding of RACK1 to the 40S subunit is crucial for this translation arrest (Kuroha et al., 2010). Translation arrest by a nascent peptide results in Dom34/Hbs1-independent endonucleolytic cleavage of mRNA, and RACK1 stimulates this cleavage event. We propose that RACK1 stimulates translation arrest induced by basic amino acid sequences that leads to endonucleolytic cleavage of the mRNA as well as to cotranslational protein degradation.

We also found that Upf1p prevents the accumulation of aberrant proteins by stimulating the degradation of specific PTC products (Kuroha et al., 2009). Taken together, we propose that protein degradation by the proteasome, in concert with rapid mRNA degradation, plays an important role in preventing the expression of abnormal proteins derived from aberrant mRNAs.

  • Tsuboi, T., Kuroha, K., Kudo, K., Makino S., Inoue, E., Kashima, I. and Inada, T.
    Dom34:Hbs1 Plays a General Role in Quality Control Systems by Dissociation of a Stalled Ribosome at the 3’ End of Aberrant mRNA
    Mol Cell 26: 518-529. (2012)
    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 Non-Stop-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.
  • Kuroha, K., Akamatsu, M., Dimitrova, L., Ito, T., Kato, Y. Shirahige, K. and Inada, T
    RACK1 stimulates nascent polypeptide-dependent translation arrest.
    EMBO Reportsl 11: 956-961. (2010)
    Nascent peptide-dependent translation arrest plays a crucial role in the quality control of eukaryotic gene expression. Here we show evidence that the receptor for activated C kinase (RACK1) participates in nascent peptide-dependent translation arrest, and that the binding of RACK1 to the 40S subunit is crucial for this translation arrest. Translation arrest by a nascent peptide results in Dom34/Hbs1-independent endonucleolytic cleavage of mRNA, and RACK1 stimulates this cleavage event. We propose that RACK1 stimulates translation arrest induced by basic amino acid sequences that leads to endonucleolytic cleavage of the mRNA as well as to cotranslational protein degradation.
  • Kobayashi, K. Kikuno, I. Kuroha, K. Saito, K. Ito, K. Ishitani, R. Inada, T. and Nureki, O.
    Structural Basis for mRNA Surveillance by Archaeal Pelota and GTP-bound EF1α Complex.
    Proc. Natl. Acad. Sci. USAl 107: 17575-17579. (2010)
    No-go decay and nonstop decay are mRNA surveillance pathways that detect translational stalling and degrade the underlying mRNA, allowing the correct translation of the genetic code. In eukaryotes, the protein complex of Pelota (yeast Dom34) and Hbs1 translational GTPase recognizes the stalled ribosome containing the defective mRNA. Recently, we found that archaeal Pelota (aPelota) associates with archaeal elongation factor 1α (aEF1α) to act in the mRNA surveillance pathway, which accounts for the lack of an Hbs1 ortholog in archaea. Here we present the complex structure of aPelota and GTP-bound aEF1α determined at 2.3-Å resolution. The structure reveals how GTP-bound aEF1α recognizes aPelota and how aPelota in turn stabilizes the GTP form of aEF1α. Combined with the functional analysis in yeast, the present results provide structural insights into the molecular interaction between eukaryotic Pelota and Hbs1. Strikingly, the aPelota·aEF1α complex structurally resembles the tRNA·EF-Tu complex bound to the ribosome. Our findings suggest that the molecular mimicry of tRNA in the distorted "A/T state" conformation by Pelota enables the complex to efficiently detect and enter the empty A site of the stalled ribosome.
  • Tsuboi, T. and Inada, T.
    Tethering of poly(A) binding protein interferes with non-translated mRNA decay from 5' end in yeast.
    J. Biol. Chem.l 285: 33589-33601. (2010)
    The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5' end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5'-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3' to 5' mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1-34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1-34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.
  • Kuroha, K., Tatematsu, T. and Inada, T.
    Upf1p stimulates proteasome-mediated degradation of the product derived from the specific nonsense-containing mRNA.
    EMBO Reportsl 10:1265-1271. (2009)
    Aberrant messenger RNAs containing a premature termination codon (PTC) are eliminated by the nonsense-mediated mRNA decay (NMD) pathway. Here, we show that a crucial NMD factor, up frameshift 1 protein (Upf1), is required for rapid proteasome-mediated degradation of an aberrant protein (PTC product) derived from a PTC-containing mRNA. Western blot and pulse-chase analyses revealed that Upf1 stimulates the degradation of specific PTC products by the proteasome. Moreover, the Upf1-dependent, proteasome-mediated degradation of the PTC product was also stimulated by mRNAs harbouring a faux 3' untranslated region (3'-UTR). These results indicate that protein stability might be regulated by an aberrant mRNA 3'-UTR.
  • Dimitrova, L., Kuroha, K., Tatematsu, T. and Inada, T.
    Nascent peptide-dependent translation arrest leads to not4p-mediated protein degradation by the proteasome.
    J. Biol. Chem.l 284: 10343-10352. (2009)
    The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stem-loop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4Delta mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5 E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome.
  • Ito-Harashima, S., Kuroha, K., Tatematsu, T. and Inada, T.
    Translation of poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast.
    Genes Dev.l 21: 519-524. (2007)
    mRNA surveillance system represses the expression of nonstop mRNA by rapid mRNA degradation and translation repression. Here we show that the level of protein product of nonstop mRNA containing a poly(A) tail was reduced 100-fold, and this reduction was due to rapid mRNA degradation, translation repression, and protein destabilization, at least in part, by the proteasome. Insertion of a poly(A) tract upstream of a termination codon resulted in translation repression and protein destabilization, but not rapid mRNA decay. We propose that translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization.
  • *Inada, T. and Aiba, H. (*corresponding author)
    Translation of aberrant mRNAs lacking a termination codon or with a shortened 3'-UTR is repressed after initiation in yeast.
    EMBO J.l 24: 1584-1595. (2005)
    A novel mRNA surveillance for mRNA lacking a termination codon (nonstop mRNA) has been proposed in which Ski7p is thought to recognize stalled ribosomes at the 3' end of mRNA. Here we report our analysis of translation and decay of nonstop mRNAs in Saccharomyces cerevisiae. Although the reduction of nonstop mRNAs was only 4.5-fold, a level that is sufficient for residual protein synthesis, translation products of nonstop mRNAs were hardly detectable. We show that nonstop mRNAs were associated with polysomes, but not with Pab1p. We also show that ribosomes translating nonstop mRNA formed stable and heavy polysome complexes with mRNA. These data suggest that ribosome stalling at the 3' end of nonstop mRNA may block further rounds of translation, hence repressing protein synthesis. Furthermore, it was found that the 5' --> 3' decay pathway was accelerated for nonstop mRNA decay in the absence of Ski7p. We also found that translation of aberrant mRNAs with a shortened 3'-UTR was repressed, suggesting that an improper spatial distance between the termination codon and the 3' end of mRNA results in translation repression.
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