Recent Progress on the Molecular Mechanism of Quality Controls Induced by Ribosome Stalling

Accurate gene expression is a prerequisite for all cellular processes. Cells actively promote correct protein folding, which prevents the accumulation of abnormal and non-functional proteins. Translation elongation is the fundamental step in gene expression to ensure cellular functions, and abnormal translation arrest is recognized and removed by the quality controls. Recent studies demonstrated that ribosome plays crucial roles as a hub for gene regulation and quality controls. Ribosome-interacting factors are critical for the quality control mechanisms responding to abnormal translation arrest by targeting its products for degradation. Aberrant mRNAs are produced by errors in mRNA maturation steps and cause aberrant translation and are eliminated by the quality control system. In this review, we focus on recent progress on two quality controls, Ribosome-associated Quality Control (RQC) and No-Go Decay (NGD), for abnormal translational elongation. These quality controls recognize aberrant ribosome stalling and induce rapid degradation of aberrant polypeptides and mRNAs thereby maintaining protein homeostasis and preventing the protein aggregation

Dual Functions of Yeast tRNA Ligase in the Unfolded Protein Response: Unconventional Cytoplasmic Splicing of HAC1 Pre-mRNA Is Not Sufficient to Release Translational Attenuation

Unconventional cytoplasmic splicing of HAC1 mRNA is essential for the yeast unfolded protein response (UPR). The UPR requires translational regulation of unspliced and spliced forms of HAC1 mRNAs. Here we report that tRNA ligase, Rlg1p, which ligates HAC1 exons in its splicing, has another face as a translational regulator of HAC1 mRNA

The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation

Ubiquitination is a major post-translational modification of ribosomal proteins. The role of ubiquitination in the regulation of ribosome functions is still being elucidated. However, the importance of ribosome deubiquitination remains unclear. Here, we show that the cycle of ubiquitination and deubiquitination of the 40S ribosome subunit eS7 is important for efficient translation. eS7 ubiquitination at lysine 83 is required for efficient protein translation. We identified Otu2 and Ubp3 as the deubiquitinating enzymes for eS7. An otu2Δubp3Δ mutation caused a defect in protein synthesis. Ubp3 inhibited polyubiquitination of eS7 in polysomes to keep eS7 in a mono-ubiquitinated form, whereas Otu2 was specifically bound to the free 40S ribosome and promoted the dissociation of mRNAs from 40S ribosomes in the recycling step. Our results provide clues for understanding the molecular mechanism of the translation system via a ubiquitination-deubiquitination cycle

Validity of strong lensing statistics for constraints on the galaxy evolution model

We examine the usefulness of the strong lensing statistics to constrain the evolution of the number density of lensing galaxies by adopting the values of the cosmological parameters determined by recent WMAP observation. For this purpose, we employ the lens-redshift test proposed by Kochanek (1992) and constrain the parameters in two evolution models, simple power-law model characterized by the power law indexes $nu_{n}$ and $\nu_{v}$ and the evolution model by Mitchell et al. (2005) based on CDM structure formation scenario. We use the well-defined lens sample from the Sloan Digital Sky Survey (SDSS) and this is similarly sized samples used in the previous studies. Furthermore, we adopt the velocity dispersion function of early-type galaxies based on SDSS DR1 and DR5. It turns out that the indexes of power-law model are consistent with the previous studies, thus our results indicate the mild evolution in the number and velocity dispersion of early-type galaxies out to z = 1. However we found that the values for p and q used by Mitchell et al. are inconsistent with the presently available observational data. More complete sample is necessary to withdraw more realistic determination on these parameters.Comment: Accepted to MNRAS, 7 pages, 3 figure

Ubiquitination of stalled ribosome triggers ribosome-associated quality control

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits

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

Co-Translational Quality Control Induced by Translational Arrest

Genetic mutations, mRNA processing errors, and lack of availability of charged tRNAs sometimes slow down or completely stall translating ribosomes. Since an incomplete nascent chain derived from stalled ribosomes may function anomalously, such as by forming toxic aggregates, surveillance systems monitor every step of translation and dispose of such products to prevent their accumulation. Over the past decade, yeast models with powerful genetics and biochemical techniques have contributed to uncovering the mechanism of the co-translational quality control system, which eliminates the harmful products generated from aberrant translation. We here summarize the current knowledge of the molecular mechanism of the co-translational quality control systems in yeast, which eliminate the incomplete nascent chain, improper mRNAs, and faulty ribosomes to maintain cellular protein homeostasis

Optimized protocol for tRNA identification in the ribosomal complexes from human cell lines

Summary: Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with Nuclear Export Mediator Factor (NEMF), a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative Polymerase chain reaction (PCR). Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers.For complete details on the use and execution of this protocol, please refer to Udagawa et al. (2021)

Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex

The colliding ribosomes are ubiquitinated by the sensor protein Hel2, leading to noncanonical subunit dissociation by the ribosome associated quality control trigger (RQT) complex. Here the authors reveal the decoding mechanism of the ribosome ubiquitin code by the RQT complex