The nascent polypeptide in the 60S subunit determines the Rqc2-dependency of ribosomal quality control.

Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.

RQT complex dissociates ribosomes collided on endogenous RQC substrate SDD1.

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.

Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways.

Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no-go decay) and the nascent polypeptide (RQC: ribosome-associated quality control). RQC requires Hel2-dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2-dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an in vitro translation reaction. Cryo-EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2-mediated cleavages upstream of the disome, governed by initial Not4-mediated monoubiquitination of eS7 and followed by Hel2-mediated K63-linked polyubiquitination. We propose that Hel2-mediated ribosome ubiquitination is required both for canonical NGD (NGDRQC+) and RQC coupled to the disome and that RQC-uncoupled NGD outside the disome (NGDRQC-) can occur in a Not4-dependent manner.

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.

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.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.

Protein quality control systems associated with no-go and nonstop mRNA surveillance in yeast.

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.

Ultrasound evaluation of cartilage damage in osteochondral lesions of the talar dome and correlation with clinical etiology: a preliminary report.

BACKGROUND: The current study aimed to determine whether the acoustic properties of living human cartilage during arthroscopy differ between damage from trauma and that from pigmented villonodular synovitis (PVNS). METHODS: Nine patients were evaluated with ultrasound during arthroscopy. As a quantitative index of cartilage quality, the percentage maximal magnitude (maximal magnitude of the measurement area divided by that of the intact cartilage; %MM) was selected. After ultrasound evaluation, the measurement points were divided into two groups on the basis of the etiologic findings (group T: cartilage damage from trauma and group P: cartilage damage from PVNS) and analyzed for the presence of significant differences in ultrasound analysis. RESULTS: In the ultrasound findings, the %MM values ranged from 34.4% to 92.3%. According to the etiologic differences, the mean %MM was 81.0% in group T and 39.3% in group P, and significantly higher in group T than in group P (p < 0.01). CONCLUSIONS: This study showed a correlation between the ultrasound results and the cartilage lesion etiology. Ultrasound evaluation may be useful for elucidating the process of articular cartilage degeneration with trauma and PVNS.

Differential acoustic properties of early cartilage lesions in living human knee and ankle joints.

OBJECTIVE: Although numerous studies have been performed to determine whether there are histologic, biochemical, biomechanical, and metabolic differences between knee and ankle cartilage, none have investigated the presence of such differences in living human cartilage. We previously developed an ultrasonic evaluation system for articular cartilage that analyzes the A-mode echogram using wavelet transformation. The current study was undertaken to determine whether the acoustic properties of living human cartilage differ between knee and ankle joints. METHODS: Twenty-eight patients were subjected to ultrasonic evaluation under arthroscopy. After arthroscopic grading, the cartilage was measured using an ultrasonic probe. Two quantitative parameters were used, i.e., the maximum magnitude and the echo duration at the 95% interval of the maximum magnitude. RESULTS: In intact cartilage, the maximum magnitude and echo duration did not differ between the knee and the ankle. In lesional cartilage, in contrast, the maximum magnitude was higher, and the echo duration was shorter, in the ankle than in the knee. These differences were statistically significant. CONCLUSION: Ultrasound findings could be used to judge the degree of early cartilage degeneration in vivo on the basis of objective data such as the maximum magnitude and echo duration. Because we were unable to quantitatively analyze the biochemical and biomechanical properties of the cartilage in this study, our biochemical and biomechanical findings are based only on qualitative assessment. Nevertheless, the results indicate that this ultrasonic evaluation system may be useful for elucidating the processes of articular cartilage degeneration in osteoarthritis.

Novel ultrasonic evaluation of tissue-engineered cartilage for large osteochondral defects--non-invasive judgment of tissue-engineered cartilage.

Although numerous methods for regenerating articular cartilage have been investigated, the regenerated tissue showed various histological findings from hyaline-like cartilage to fibrous tissue. Without biopsy, we are unable to know whether the cartilage regeneration method was histologically successful or not. We developed a new ultrasonic evaluation system for articular cartilage using the maximum magnitude (MM) from ultrasonic analysis. The purpose of this study was to investigate the usefulness of ultrasonic judgment of the cartilage regeneration procedure. Using our system we quantitatively evaluated tissue-engineered cartilage in rabbit cartilage defects. The specimens were retrospectively divided into two groups on the basis of histological findings and investigated whether significant differences in ultrasonic analysis could be found between the two (group H: hyaline-like cartilage group, successful; group F: fibrous tissue group, failure). In the ultrasonic findings, the MM was 1.11+/-0.32 in group H and 0.65+/-0.18 in group F and these differences were significant (P=0.00061). Our results suggest that the ultrasonic evaluation system used in the present study is capable of judging the success or failure of cartilage regeneration procedures, and therefore, it could be a valuable tool arthroscopic diagnosis of cartilage regeneration.

Quantitative ultrasound can assess the regeneration process of tissue-engineered cartilage using a complex between adherent bone marrow cells and a three-dimensional scaffold.

Articular cartilage (hyaline cartilage) defects resulting from traumatic injury or degenerative joint disease do not repair themselves spontaneously. Therefore, such defects may require novel regenerative strategies to restore biologically and biomechanically functional tissue. Recently, tissue engineering using a complex of cells and scaffold has emerged as a new approach for repairing cartilage defects and restoring cartilage function. With the advent of this new technology, accurate methods for evaluating articular cartilage have become important. In particular, in vivo evaluation is essential for determining the best treatment. However, without a biopsy, which causes damage, articular cartilage cannot be accurately evaluated in a clinical context. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the cartilage are measured by introducing an ultrasonic probe during arthroscopy of the knee joint. The purpose of the current study was to determine the efficacy of this ultrasound system for evaluating tissue-engineered cartilage in an experimental model involving implantation of a cell/scaffold complex into rabbit knee joint defects. Ultrasonic echoes from the articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the percentage maximum magnitude (the maximum magnitude of the measurement area of the operated knee divided by that of the intact cartilage of the opposite, nonoperated knee; %MM) was used as a quantitative index of cartilage regeneration. Using this index, the tissue-engineered cartilage was examined to elucidate the relations between ultrasonic analysis and biochemical and histological analyses. The %MM increased over the time course of the implant and all the hyaline-like cartilage samples from the histological findings had a high %MM. Correlations were observed between the %MM and the semiquantitative histologic grading scale scores from the histological findings. In the biochemical findings, the chondroitin sulfate content increased over the time course of the implant, whereas the hydroxyproline content remained constant. The chondroitin sulfate content showed a similarity to the results of the %MM values. Ultrasonic measurements were found to predict the regeneration process of the tissue-engineered cartilage as a minimally invasive method. Therefore, ultrasonic evaluation using a wavelet map can support the evaluation of tissue-engineered cartilage using cell/scaffold complexes.

Mechanical effects of autogenous osteochondral surgical grafting procedures and instrumentation on grafts of articular cartilage.

PURPOSE: To analyze the mechanical effects of autogenous osteochondral grafting procedures on articular cartilage. METHODS: The intensity, duration, and interval (indexes of stiffness, surface irregularity, and thickness) of the cartilage were assessed in a porcine model using an ultrasonic measurement system. In 7 of 12 knees, 6-mm-diameter plugs were harvested from the donor knees and grafted into 5-mm recipient holes at 3 different points per knee (21 plugs). In the remaining 5 knees, 5-mm plugs were harvested and returned to their original position (28 plugs). RESULTS: No significant differences in the intensity, duration, and interval of the cartilage were observed with the plugs before harvesting and after grafting by the paired t test. The 3 indexes of the 6- and 5-mm plugs that were grafted correlated significantly with those before they were. CONCLUSIONS: These results suggest that osteochondral graft surgery does not affect the stiffness, surface irregularity, and thickness of the cartilage of the plugs at time zero.

Measurement of the mechanical condition of articular cartilage with an ultrasonic probe: quantitative evaluation using wavelet transformation.

OBJECTIVE: To develop a new diagnostic technology to evaluate articular cartilage quantitatively by introducing an ultrasonic probe into the knee joint under arthroscopy and analyzing the A-mode echogram by means of wavelet transformation. DESIGN: Quantitative evaluation using comparison of two indices on the wavelet map and macroscopic evaluation using the Outerbridge classification. As the quantitative indices on the wavelet map, the maximum magnitude and the echo duration which was defined as the length of time that included 95% of echo signal were selected. BACKGROUND: Quantitative evaluation of articular cartilage in situ is required for new tissue-engineered cartilage but an evaluation system that fully meets this requirement has yet to be established for clinical use. METHODS: Human articular cartilage specimens were analyzed using an ultrasonic probe after macroscopic evaluation and the cartilage characteristics on the echo duration-maximum magnitude graph were examined. RESULTS: There were significant differences between grade 1 and 3, and grade 2 and 3 in the echo duration and maximum magnitude. The cartilage specimens had a L-shaped distribution in echo duration-maximum magnitude graph. CONCLUSIONS: The present study is the new quantitative evaluation system of the articular cartilage in situ and a clinical trial under arthroscopy is presently underway. The ultrasonic measurement is highly reproducible, so we can expect this system to be suitable for in situ reliable examination under arthroscopy. RELEVANCE: Precise evaluation of articular cartilage is of particular importance for longitudinal clinical trials for determination of best surgical option or effect of new chondroprotective drugs in arthritic disease and rheumatoid arthritis.