Toshifumi Inada

A Ribosome-Bound Quality Control Complex Triggers Degradation of Nascent Peptides and Signals Translation Stress

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Mechanism responsible for glucose–lactose diauxie in Escherichia coli: challenge to the cAMP model

Background : The inhibition of β‐galactosidase expression in glucose–lactose diauxie is a typical example of the glucose effect in Escherichia coli. It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level. However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose–lactose system.

Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome

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 not4Δ 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.

Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli

The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E‐dependent manner in response to the accumulation of glucose 6‐P or fructose 6‐P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C‐terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E‐based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.

SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon

SsrA RNA of Escherichia coli, also known as 10Sa RNA or tmRNA, acts both as tRNA and mRNA when ribosomes are paused at the 3′ end of an mRNA lacking a stop codon. This process, referred to as trans‐translation, leads to the addition of a short peptide tag to the C‐terminus of the incomplete nascent polypeptide. The tagged polypeptide is then degraded by C‐terminal‐specific proteases. Here, we focused on endogenous targets for the SsrA system and on a potential regulatory role of SsrA RNA. First, we show that trans‐translation events occur frequently in normally growing E.coli cells. More specifically, we report that the lacI mRNA encoding Lac repressor (LacI) is a specific natural target for trans‐translation. The binding of LacI to the lac operators results in truncated lacI mRNAs that are, in turn, recognized by the SsrA system. The SsrA‐mediated tagging and proteolysis of LacI appears to play a role in cellular adaptation to lactose availability by supporting a rapid induction of lac operon expression.

Expression of the glucose transporter gene, ptsG, is regulated at the mRNA degradation step in response to glycolytic flux in Escherichia coli

We report a novel post‐transcriptional control of the ptsG gene encoding the major glucose transporter IICBGlc. We demonstrate that the level of IICBGlc is markedly reduced when the glycolytic pathway is blocked by a mutation in either the pgi or pfkA gene encoding phosphoglucose isomerase or phosphofructokinase, respectively. This down‐regulation of ptsG is not exerted at the transcriptional level. Both northern blot and S1 analyses demonstrate that the mutation dramatically accelerates the degradation of ptsG mRNA. The degradation of ptsG mRNA occurs in wild‐type cells when α‐methylglucoside, a non‐ metabolizable analog of glucose, is present in the medium. The addition of any one of the glycolytic intermediates downstream of the block prevents the degradation of ptsG mRNA. The rapid degradation of ptsG mRNA is eliminated when RNase E is thermally inactivated. We conclude that the glycolytic pathway controls ptsG expression by modulating RNase E‐mediated mRNA degradation. This is the first instance in which the glycolytic flux has been shown to affect the expression of a specific gene through mRNA stability.

Translation of aberrant mRNAs lacking a termination codon or with a shortened 3′‐UTR is repressed after initiation in yeast

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.

One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.

We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNAs translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.

Receptor for activated C kinase 1 stimulates nascent polypeptide‐dependent translation arrest

Nascent peptide‐dependent translation arrest is crucial for the quality control of eukaryotic gene expression. Here we show that the receptor for activated C kinase 1 (RACK1) participates in nascent peptide‐dependent translation arrest, and that its binding to the 40S subunit is crucial for this. Translation arrest by a nascent peptide results in Dom34/Hbs1‐independent endonucleolytic cleavage of mRNA, and this is stimulated by RACK1. We propose that RACK1 stimulates the translation arrest that is induced by basic amino‐acid sequences that leads to endonucleolytic cleavage of the mRNA, as well as to co‐translational protein degradation.

A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli

Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double‐negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP–cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP–cAMP. DNase I footprinting experiments revealed that CRP–cAMP binds at two sites centred at −40.5 and −95.5 and that Mlc binds at two regions centred around −8 and −175. The binding of CRP–cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP–cAMP. Gel‐mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.