Publisher Correction: Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.

Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1-6. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails)7-9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10-12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.

Translation arrest as a protein quality control system for aberrant translation of the 3'-UTR in mammalian cells.

Read-through or mutations of a stop codon resulting in translation of the 3'-UTR produce potentially toxic C-terminally extended proteins. However, quality control mechanisms for such proteins are poorly understood in mammalian cells. Here, a comprehensive analysis of the 3'-UTRs of genes associated with hereditary diseases identified novel arrest-inducing sequences in the 3'-UTRs of 23 genes that can repress the levels of their protein products. In silico analysis revealed that the hydrophobicity of the polypeptides encoded in the 3'-UTRs is correlated with arrest efficiency. These results provide new insight into quality control mechanisms mediated by 3'-UTRs to prevent the production of C-terminally extended cytotoxic proteins.

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.

Cytosolic Protein Vms1 Links Ribosome Quality Control to Mitochondrial and Cellular Homeostasis.

Eukaryotic cells have evolved extensive protein quality-control mechanisms to remove faulty translation products. Here, we show that yeast cells continually produce faulty mitochondrial polypeptides that stall on the ribosome during translation but are imported into the mitochondria. The cytosolic protein Vms1, together with the E3 ligase Ltn1, protects against the mitochondrial toxicity of these proteins and maintains cell viability under respiratory conditions. In the absence of these factors, stalled polypeptides aggregate after import and sequester critical mitochondrial chaperone and translation machinery. Aggregation depends on C-terminal alanyl/threonyl sequences (CAT-tails) that are attached to stalled polypeptides on 60S ribosomes by Rqc2. Vms1 binds to 60S ribosomes at the mitochondrial surface and antagonizes Rqc2, thereby facilitating import, impeding aggregation, and directing aberrant polypeptides to intra-mitochondrial quality control. Vms1 is a key component of a rescue pathway for ribosome-stalled mitochondrial polypeptides that are inaccessible to ubiquitylation due to coupling of translation and translocation.

Isolation of Mitochondria from Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is an important model organism to study cellular structure and function. Due to its excellent accessibility to genetics and biochemical and microscopic analyses, studies with yeast have provided fundamental insights into mitochondrial biology. Yeast offers additional advantages because it can grow under fermenting conditions when oxidative phosphorylation is not obligatory and because the majority of mitochondrial structure and function are largely conserved during evolution. Isolation of mitochondria is an important technique for mitochondrial studies. This chapter focuses on procedures for the isolation and purification of intact yeast mitochondria that can be used for numerous functional assays as well as for analyses of mitochondrial ultrastructure.

An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation.

Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution.

Quality control of nonstop membrane proteins at the ER membrane and in the cytosol.

Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions.

No evidence for AID/MBD4-coupled DNA demethylation in zebrafish embryos.

The mechanisms responsible for active DNA demethylation remain elusive in Metazoa. A previous study that utilized zebrafish embryos provided a potent mechanism for active demethylation in which three proteins, AID, MBD4, and GADD45 are involved. We recently found age-dependent DNA hypomethylation in zebrafish, and it prompted us to examine if AID and MBD4 could be involved in the phenomenon. Unexpectedly, however, we found that most of the findings in the previous study were not reproducible. First, the injection of a methylated DNA fragment into zebrafish eggs did not affect either the methylation of genomic DNA, injected methylated DNA itself, or several loci tested or the expression level of aid, which has been shown to play a role in demethylation. Second, aberrant methylation was not observed at certain CpG islands following the injection of antisense morpholino oligonucleotides against aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA lacked a coding region for the methyl-CpG binding domain, which was assumed to be necessary for guidance to target regions. Taken together, we concluded that there is currently no evidence to support the proposed roles of AID and MBD4 in active demethylation in zebrafish embryos.

Aim24 and MICOS modulate respiratory function, tafazzin-related cardiolipin modification and mitochondrial architecture.

Structure and function of mitochondria are intimately linked. In a search for components that participate in building the elaborate architecture of this complex organelle we have identified Aim24, an inner membrane protein. Aim24 interacts with the MICOS complex that is required for the formation of crista junctions and contact sites between inner and outer membranes. Aim24 is necessary for the integrity of the MICOS complex, for normal respiratory growth and mitochondrial ultrastructure. Modification of MICOS subunits Mic12 or Mic26 by His-tags in the absence of Aim24 leads to complete loss of cristae and respiratory complexes. In addition, the level of tafazzin, a cardiolipin transacylase, is drastically reduced and the composition of cardiolipin is modified like in mutants lacking tafazzin. In conclusion, Aim24 by interacting with the MICOS complex plays a key role in mitochondrial architecture, composition and function. DOI: http://dx.doi.org/10.7554/eLife.01684.001.

Decrease in cytosine methylation at CpG island shores and increase in DNA fragmentation during zebrafish aging.

Age-related changes in DNA methylation have been demonstrated in mammals, but it remains unclear as to the generality of this phenomenon in vertebrates, which is a criterion for the fundamental cause of senescence. Here we showed that the zebrafish genome gradually and clearly lost methylcytosine in somatic cells, but not in male germ cells during aging, and that age-dependent hypomethylation preferentially occurred at a particular domain called the CpG island shore, which is associated with vertebrates' genes and has been shown to be hypomethylated in humans with age. We also found that two CpG island shores hypomethylated in zebrafish oocytes were de novo methylated in fertilized eggs, which suggests that the zebrafish epigenome is reset upon fertilization, enabling new generations to restart with a heavily methylated genome. Furthermore, we observed an increase in cleavage of the zebrafish genome to an oligonucleosome length in somatic cells from the age of 12 months, which is suggestive of an elevated rate of apoptosis in the senescent stage.

Roles of dom34:hbs1 in nonstop protein clearance from translocators for normal organelle protein influx.

Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane. These stuck nonstop secretory proteins avoid proteasomal degradation in the cytosol, but are instead released into the ER lumen through stalled ribosome and translocator channels by Dom34:Hbs1. We also found that nonstop mitochondrial proteins are cleared from the mitochondrial translocator, the TOM40 complex, by Dom34:Hbs1. Clearance of stuck nonstop proteins from organellar translocator channels is crucial for normal protein influx into organelles and for normal cell growth, especially when nonstop mRNA decay does not function efficiently.

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation.

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.

Rotatory laxity evaluation of the knee using modified Slocum's test in open magnetic resonance imaging.

PURPOSE: The authors have developed a quantitative method to evaluate rotatory laxity of the anterior cruciate ligament (ACL)-deficient knee using an open MRI. In this study, we evaluated the correlation between the pivot-shift test grading and values measured with this method. Furthermore, the articular contact area in the lateral compartment during the pivot shift was examined. METHODS: Ninety-two subjects were evaluated in this study. Among them, 36 subjects were evaluated before the ACL reconstruction, and 56 subjects were evaluated 1 year after the ACL reconstruction. Subjects were placed in the open MRI scanner in the posture based on the Slocum's ALRI test. Anterior translation of the tibia was measured in the lateral and medial compartments in the sagittal images of the MRI. The correlation between the grading of the pivot-shift test and either anterior translation of the lateral compartment, or the difference between translation of the medial compartment and the lateral compartment, was evaluated by logistic analysis. Furthermore, we examined the contact area of the articular surface in the lateral compartment with variation in the pivot-shift grading. RESULTS: The anterior translation of the lateral compartment had a significant correlation with the pivot-shift grading. A discriminant analysis showed that the lateral translation could predict the grade of the pivot-shift test more accurately than the lateral minus medial translation (84.8% vs. 70.7%, P < 0.001). The summit of the convex lateral tibial articular surface is located anterior to the closest point between the lateral femoral condyle and the tibial plateau in the pivot-shift-positive subjects, while it is located posterior to the femoral condyle in the pivot-shift-negative subjects. CONCLUSION: The anterior translation in the lateral compartment measured with this method allows objective and quantitative evaluation of the rotatory laxity that causes the pivot-shift phenomenon. The grade of the pivot-shift test is determined by the magnitude of the movement of the lateral compartment when the femoral condyle surmounts the convex tibial articular surface, rather than by the deviation of the rotational angle of the tibia. LEVEL OF EVIDENCE: II.

Evaluation of cartilage degradation in arthritis using T1ρ magnetic resonance imaging mapping.

T1ρ magnetic resonance imaging (MRI) can be used to map proteoglycan (PG) loss in cartilage. Here, we used T1ρ MRI to map cartilage degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). Tissue samples were obtained from five RA patients and 14 OA patients following total knee arthroplasty (TKA). Three parameters were measured: First, macroscopic grading of cartilage sample tissues was performed on a 5-grade scale (G0: normal, G1: swelling, G2: superficial fibrillation, G3: deep fibrillation, G4: subchondral bone exposure). Second, semi-quantitative values of PG were assessed by measuring the optical density of Safranin-O-stained paraffin sections that had been digitally photographed. Third, cartilage was divided into superficial and deep layers and the T1ρ values were quantified. T1ρ values of OA and RA in the superficial layers showed significant differences between groups (G0/1 and G0/2 for OA; G0/2 and G1/2 for RA). In the deep layers, T1ρ values of OA and RA also differed significantly between groups. In both the superficial and deep layers, there was a significant correlation between the mean T1ρ values and macroscopic grading (P < 0.01 for OA, P < 0.001 for RA). We found a negative correlation between the score of Safranin-O staining and T1ρ values (r = -0.61 for OA, r = -0.79 for RA). In addition, RA subjects had significantly higher T1ρ values than OA subjects of similar morphologic grade. In conclusion, T1ρ MRI is able to detect and map the early stages of cartilage degradation in OA and RA. This method is reliable and useful for the evaluation of macromolecular changes in arthritic cartilage.

New suture method for radial tears of the meniscus: biomechanical analysis of cross-suture and double horizontal suture techniques using cyclic load testing.

BACKGROUND: Repair of complete radial meniscal tears is a key to restoring the mechanical integrity necessary to maintain hoop tension in the meniscus. The primary stability of the meniscal repair is one of the most important factors for meniscal healing, but the biomechanical structural properties of different repair techniques for complete radial meniscal tears remain unknown. HYPOTHESIS: Our novel cross-suture technique with suturing oblique to the collagen fibrils of the meniscus will yield better fixation than the standard double horizontal suture technique with suturing parallel to the collagen fibrils in the meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Biomechanical investigation was performed on 40 fresh human menisci (2 groups of 20 menisci each) from patients who underwent total knee arthroplasty. In the cross-suture technique group (group A), the sutures crossed over 5 mm from the tear and 5 mm and 10 mm from the rim. In the double horizontal suture technique group (group B), the sutures were parallel and had the same attachment points as group A. The specimens were cyclically loaded 500 times between 5 and 30 N and then loaded to failure after completion of the cyclic load testing. RESULTS: Compared with the double horizontal suture group, the cross-suture group had a significantly higher ultimate failure load (78.96 ± 19.27 N vs 68.16 ± 12.92 N; P < .05), significantly greater stiffness (8.01 ± 1.54 N/mm vs 6.46 ± 1.12 N/mm; P < .05), and significantly lower displacement (5.74 ± 1.84 mm vs 8.56 ± 2.39 mm; P < .05) after a 500-cycle loading protocol. CONCLUSION: Our cross-suture technique significantly improved the structural properties of the repaired complete radial meniscal tears. CLINICAL RELEVANCE: The cross-suture technique for repair of radial meniscal tears provides high stability and could be a promising solution in young and in active patients.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon.

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis.

Comparison of rotatory stability after anterior cruciate ligament reconstruction between single-bundle and double-bundle techniques.

BACKGROUND: Controversy persists as to whether double-bundle reconstruction of the anterior cruciate ligament (ACL) has any clinical advantage over single-bundle reconstruction. Several studies have used subjective and nonquantitative manual tests to evaluate the rotatory stability of the knee. The authors have developed a method to quantitate the rotatory stability of the ACL-deficient knee using open magnetic resonance imaging (MRI). HYPOTHESIS: Anatomic double-bundle reconstruction restores rotatory stability significantly better than does single-bundle reconstruction. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Twenty-three consecutive patients treated with the single-bundle reconstruction (group S) and 25 consecutive patients treated with the anatomic double-bundle reconstruction (group D) were evaluated. Both reconstruction procedures were performed using hamstring tendon autografts. The Slocum anterolateral rotatory instability (ALRI) test was performed 1 year after surgery using open MRI. To assess rotatory stability, we measured the difference in anterior tibial translation between medial and lateral compartments in the sagittal plane and defined this difference as the ALRI value. In addition, clinical examinations consisting of the Lysholm knee score, Tegner activity score, KT-2000 arthrometer anterior translation examination, and the pivot-shift test were carried out. RESULTS: The mean side-to-side difference in ALRI values was significantly less (P < .001) in double-bundle reconstruction (mean, 1.2 mm) than in single-bundle reconstruction (mean, 4.1 mm). The mean side-to-side difference in KT-2000 arthrometer measurements was significantly less (P = .014) in double-bundle reconstruction (mean, 1.2 mm) than in single-bundle reconstruction (mean, 2.6 mm). The difference in the incidence of positive pivot-shift tests between group S (43%) and group D (16%) did not reach the level of statistical significance (P = .058). No significant differences in Lysholm score or Tegner score between the groups were observed. CONCLUSION: The rotatory stability of anatomic double-bundle reconstruction was significantly better than the rotatory stability of single-bundle reconstruction.

Extensive expansion and diversification of the chemokine gene family in zebrafish: identification of a novel chemokine subfamily CX.

BACKGROUND: The chemokine family plays important roles in cell migration and activation. In humans, at least 44 members are known. Based on the arrangement of the four conserved cysteine residues, chemokines are now classified into four subfamilies, CXC, CC, XC and CX3C. Given that zebrafish is an important experimental model and teleost fishes constitute an evolutionarily diverse group that forms half the vertebrate species, it would be useful to compare the zebrafish chemokine system with those of mammals. Prior to this study, however, only incomplete lists of the zebrafish chemokine genes were reported. RESULTS: We systematically searched chemokine genes in the zebrafish genome and EST databases, and identified more than 100 chemokine genes. These genes were CXC, CC and XC subfamily members, while no CX3C gene was identified. We also searched chemokine genes in pufferfish fugu and Tetraodon, and found only 18 chemokine genes in each species. The majority of the identified chemokine genes are unique to zebrafish or teleost fishes. However, several groups of chemokines are moderately similar to human chemokines, and some chemokines are orthologous to human homeostatic chemokines CXCL12 and CXCL14. Zebrafish also possesses a novel species-specific subfamily consisting of five members, which we term the CX subfamily. The CX chemokines lack one of the two N-terminus conserved cysteine residues but retain the third and the fourth ones. (Note that the XC subfamily only retains the second and fourth of the signature cysteines residues.) Phylogenetic analysis and genome organization of the chemokine genes showed that successive tandem duplication events generated the CX genes from the CC subfamily. Recombinant CXL-chr24a, one of the CX subfamily members on chromosome 24, showed marked chemotactic activity for carp leukocytes. The mRNA was expressed mainly during a certain period of the embryogenesis, suggesting its role in the zebrafish development. CONCLUSION: The phylogenic and genomic organization analyses suggest that a substantial number of chemokine genes in zebrafish were generated by zebrafish-specific tandem duplication events. During such duplications, a novel chemokine subfamily termed CX was generated in zebrafish. Only two human chemokines CXCL12 and CXCL14 have the orthologous chemokines in zebrafish. The diversification observed in the numbers and sequences of chemokines in the fish may reflect the adaptation of the individual species to their respective biological environment.

Fgf signaling negatively regulates Nodal-dependent endoderm induction in zebrafish.

In zebrafish development, Nodal signaling is critical for the induction of endoderm and mesoderm. Three transcription factors downstream of Nodal, Bonnie and Clyde (Bon), Faust (Fau)/Gata5 and Casanova (Cas), are required for endoderm induction. However, it is not yet fully understood how the Nodal signaling pathway regulates the decision process of endoderm and mesoderm induction. In this study, we focused on Fgf signaling, downstream of Nodal signaling, during endoderm induction. We found that activation of Fgf signaling decreases the number of cas-expressing endodermal cells. Conversely, inhibition of this signaling increases the number of endodermal cells without affecting the expression of Nodal, Nodal antagonists, bon or fau/gata5. Inhibition of Fgf signaling in endoderm mutants suggests that this signaling negatively regulates cas expression by a pathway parallel to Bon and Fau/Gata5 in the molecular cascade leading to endoderm. Furthermore, activation of Fgf signaling can overcome Cas-mediated abrogation of mesodermal gene expression. Altogether, these results suggest that Fgf signaling negatively regulates endoderm induction, possibly through repression of cas expression and down-regulation of Cas function.