Release of Ubiquitinated and Non-ubiquitinated Nascent Chains from Stalled Mammalian Ribosomal Complexes by ANKZF1 and Ptrh1.

The ribosome-associated quality control (RQC) pathway degrades nascent chains (NCs) arising from interrupted translation. First, recycling factors split stalled ribosomes, yielding NC-tRNA/60S ribosome-nascent chain complexes (60S RNCs). 60S RNCs associate with NEMF, which recruits the E3 ubiquitin ligase Listerin that ubiquitinates NCs. The mechanism of subsequent ribosomal release of Ub-NCs remains obscure. We found that, in non-ubiquitinated 60S RNCs and 80S RNCs formed on non-stop mRNAs, tRNA is not firmly fixed in the P site, which allows peptidyl-tRNA hydrolase Ptrh1 to cleave NC-tRNA, suggesting the existence of a pathway involving release of non-ubiquitinated NCs. Association with NEMF and Listerin and ubiquitination of NCs results in accommodation of NC-tRNA, rendering 60S RNCs resistant to Ptrh1 but susceptible to ANKZF1, which induces specific cleavage in the tRNA acceptor arm, releasing proteasome-degradable Ub-NCs linked to four 3'-terminal tRNA nucleotides. We also found that TCF25, a poorly characterized RQC component, ensures preferential formation of the K48-ubiquitin linkage.

Ribosomal collision is not a prerequisite for ZNF598-mediated ribosome ubiquitination and disassembly of ribosomal complexes by ASCC.

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.

Tsga8 is required for spermatid morphogenesis and male fertility in mice.

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

Two classes of EF1-family translational GTPases encoded by giant viruses.

Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin.

Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA.

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 nonstop 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.

The Upf factor complex interacts with aberrant products derived from mRNAs containing a premature termination codon and facilitates their proteasomal degradation.

Up-frameshift (Upf) factors eliminate aberrant mRNAs containing a specific premature termination codon (PTC). Here, we show that Upf complex facilitates the ubiquitin-dependent degradation of products derived from mRNA containing specific PTCs in Saccharomyces cerevisiae. The efficiency of recruitment of the Upf complex to a PTC product was correlated with the decay of the PTC product. Upf factors promoted the degradation of the human von Hippel-Lindau (VHL) protein, which is an unfolded protein in yeast cells, in a manner that depends on the presence of a faux 3'-UTR. Mass spectrometric analysis and Western blot analysis revealed that Hsp70 was associated with the PTC product. These findings suggest that the Upf complex may be recruited to ribosomes in a faux 3'-UTR-dependent manner and then associates with aberrant products to facilitate their degradation by the proteasome.

Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1α complex.

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.

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.

Upf1 stimulates degradation of the product derived from aberrant messenger RNA containing a specific nonsense mutation by the proteasome.

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.

Analysis of nonstop mRNA translation in the absence of tmRNA in Escherichia coli.

tmRNA, a product of ssrA gene, plays a crucial role in the quality control system that eliminates aberrant products of nonstop mRNAs in prokaryotes. Although tmRNA recycles ribosomes stalled at the 3' end of nonstop mRNAs, the fate of ribosomes that stall at the 3' end in the absence of tmRNA has not been extensively examined. Here we report our analysis of the translation status of nonstop mRNAs. Polysome analysis showed that nonstop mRNAs were translated efficiently, and peptidyl-tRNA was not found in any fraction in a DeltassrA strain. In vitro translation experiments using PURESYSTEM revealed that ribosomes translating nonstop mRNAs were dissociated from the 3' end of mRNA, and the peptidyl-tRNA was only weakly hydrolyzed in the monosome. These results suggest that the peptidyl-tRNA of a nonstop mRNA is hydrolyzed by an unknown factor(s) in vivo, thereby allowing a nonstop mRNA to be translated as efficiently as a normal mRNA. Possible factors involved in the hydrolysis of the peptidyl-tRNAs of nonstop mRNAs are discussed.

Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice.

Respiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

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.

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 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.

Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast.

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.