Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control.

Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation. Here we present ten cryogenic electron microscopy (cryo-EM) structures, revealing the mechanistic basis of individual steps of the CAT tailing cycle covering initiation, decoding, peptidyl transfer, and tRNA translocation. We discovered eIF5A as a crucial eukaryotic RQC factor enabling peptidyl transfer. Moreover, we observed dynamic behavior of RQC factors and tRNAs allowing for processivity of the CAT tailing cycle without additional energy input. Together, these results elucidate key differences as well as common principles between CAT tailing and canonical translation.

Sensing of individual stalled 80S ribosomes by Fap1 for nonfunctional rRNA turnover.

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

Two modes of Cue2-mediated mRNA cleavage with distinct substrate recognition initiate no-go decay.

Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGDRQC+) or RQC uncoupled (NGDRQC-). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGDRQC- but not for NGDRQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGDRQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGDRQC-), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGDRQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.

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.

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.

Identification of a novel endogenous long non-coding RNA that inhibits selenoprotein P translation.

Selenoprotein P (SELENOP) is a major plasma selenoprotein that contains 10 Sec residues, which is encoded by the UGA stop codon. The mRNA for SELENOP has the unique property of containing two Sec insertion sequence (SECIS) elements, which is located in the 3' untranslated region (3'UTR). Here, we coincidentally identified a novel gene, CCDC152, by sequence analysis. This gene was located in the antisense region of the SELENOP gene, including the 3'UTR region in the genome. We demonstrated that this novel gene functioned as a long non-coding RNA (lncRNA) that decreased SELENOP protein levels via translational rather than transcriptional, regulation. We found that the CCDC152 RNA interacted specifically and directly with the SELENOP mRNA and inhibited its binding to the SECIS-binding protein 2, resulting in the decrease of ribosome binding. We termed this novel gene product lncRNA inhibitor of SELENOP translation (L-IST). Finally, we found that epigallocatechin gallate upregulated L-IST in vitro and in vivo, to suppress SELENOP protein levels. Here, we provide a new regulatory mechanism of SELENOP translation by an endogenous long antisense ncRNA.

Crucial role of leaky initiation of uORF3 in the downregulation of HNT1 by ER stress.

eIF2α phosphorylation-mediated translational regulation is crucial for global repression of translation by various stresses, including the unfolded protein response (UPR) in eukaryotes. Although translational control during UPR has not been extensively investigated in S. cerevisiae, Hac1-mediated production of long transcripts containing uORFs was shown to repress the translation of histidine triad nucleotide-binding 1 (HNT1) mRNA. The present study showed that uORF3 is required for HNT1 expression, as well as down-regulating HNT1 translation. Translation initiation by uORF3 is inefficient, with uORF3 having a strong Kozak sequence efficiently repressing the translation of HNT1. We propose that leaky scanning of uORF3 is responsible for the downregulation of HNT1 during UPR.

Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export.

Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes. Nuclear export is a key, irreversible step in pre-ribosome maturation, but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved Saccharomyces cerevisiae GTPase Nug2 (also known as Nog2, and as NGP-1, GNL2 or nucleostemin 2 in human) has a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor. Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding before Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodelling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K(+)-dependent GTPase activity and the remodelling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery.

[A Ruptured Aneurysm at the Origin of a Duplicated Middle Cerebral Artery, Treated by Coil Embolization:A Case Report].

Duplication of the middle cerebral artery(MCA)is an anatomical variant of the MCA, originating from the distal portion of the internal carotid artery(ICA)and supplying blood flow to the tip of the temporal lobe. Cerebral aneurysms rarely develop at the bifurcation of the ICA and the duplicated MCA, but when they do develop, they may result in subarachnoid hemorrhage. We treated a 41-year-old man, who was urgently brought to our hospital because of severe headache. A computed tomography(CT)scan showed subarachnoid hemorrhage due to the rupture of an aneurysm at the origin of the duplicated MCA. The aneurysm was small and projected laterally, and coil embolization was performed employing a balloon catheter. The neck of the aneurysm was not embolized to preserve the origin of the duplicated MCA. The patient had an uneventful postoperative course, and he returned to his usual daily activities. Coil embolization is rapidly developing for treatment of cerebral aneurysms and may be the first-line treatment for duplicated MCA aneurysms. Owing to the relatively small size of such aneurysms, the risk of intraprocedural rupture should be considered, and a carefully performed balloon-assisted procedure is recommended.

[A Case of Metastatic Brain Tumor Complicated by Multiple Repeated Hemorrhages].

Metastatic brain tumor occasionally results in multiple cerebral hemorrhages. Here, we report a case of metastatic brain tumor complicated by multiple repeated cerebral hemorrhages. An 80-year-old man with a history of removal of lung cancer was admitted to our hospital because of disturbed consciousness following headache. A brain CT revealed a mass lesion of 40-mm diameter in the left cerebellum and a mass lesion of 2-mm diameter in the right temporal lobe. The cerebellar mass lesion showed homogeneous iso-density, indicating a subacute phase hemorrhage. Two days later, a follow-up CT revealed that the mass lesion had become high-density, indicating complication by a fresh hemorrhage. Hematoma was removed and histological examination was performed on the cerebellar lesion. There was leakage of dark-red liquid intraoperatively, and histological examination revealed an adenocarcinoma, indicating metastasis of the lung cancer. Additionally, necrosis and hemorrhage were identified. Postoperatively, whole brain irradiation was performed. The right temporal lesion gradually enlarged but disappeared after irradiation. In conclusion, multiple brain metastases may result in multiple cerebral hemorrhages. Repeated hemorrhage from necrosis of the carcinoma causes a slowly growing hematoma.

[A Case of Vertebral Artery Stenosis Presenting with Progressing Stroke and Treated by Percutaneous Transluminal Angioplasty].

A 75-year-old man with a history of diabetes mellitus and hypertension was suffered from dizziness and vomiting and brought to the near-by hospital. MRI showed cerebellar infarction due to right vertebral artery stenosis. Despite best medical treatment, the infarction progressed day by day and he was transferred to our hospital five days later. Neurological examination showed mild disturbance of consciousness and right hemiparesis. Right vertebral angiography revealed high-grade stenosis accompanied with atherosclerosis at the V3-V4 portion. Percutaneous transluminal angioplasty (PTA) was performed with 2.5 mm×14 mm balloon with 6 atm dilation. Postoperative course was uneventful and no further stroke occurred after the treatment. PTA was effective for vertebral artery stenosis manifested with progressing stroke. The indication of stent placement for the cerebral artery should be prudent.

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.

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

The collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.

Structural basis for clearing of ribosome collisions by the RQT complex.

Translation of aberrant messenger RNAs can cause stalling of ribosomes resulting in ribosomal collisions. Collided ribosomes are specifically recognized to initiate stress responses and quality control pathways. Ribosome-associated quality control facilitates the degradation of incomplete translation products and requires dissociation of the stalled ribosomes. A central event is therefore the splitting of collided ribosomes by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryogenic electron microscopy of RQT-ribosome complexes reveals that RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose that the Ski2-like helicase 1 (Slh1) subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the small ribosomal subunit, ultimately resulting in subunit dissociation. Our findings provide conceptual framework for a helicase-driven ribosomal splitting mechanism.

A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation.

Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.

The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway.

Ribosome collision because of translational stalling is recognized as a problematic event in translation by the E3 ubiquitin ligase Hel2, leading to non-canonical subunit dissociation followed by targeting of the faulty nascent peptides for degradation. Although Hel2-mediated quality control greatly contributes to maintenance of cellular protein homeostasis, its physiological role in dealing with endogenous substrates remains unclear. This study utilizes genome-wide analysis, based on selective ribosome profiling, to survey the endogenous substrates for Hel2. This survey reveals that Hel2 binds preferentially to the pre-engaged secretory ribosome-nascent chain complexes (RNCs), which translate upstream of targeting signals. Notably, Hel2 recruitment into secretory RNCs is elevated under signal recognition particle (SRP)-deficient conditions. Moreover, the mitochondrial defects caused by insufficient SRP are enhanced by hel2 deletion, along with mistargeting of secretory proteins into mitochondria. These findings provide insights into risk management in the secretory pathway that maintains cellular protein homeostasis.

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.

[A case of cerebral amyloid angiopathy in which a restricted subarachnoid hemorrhage recurred in the cortical sulcus following a subcortical hemorrhage].

Cerebral amyloid angiopathy (CAA) is predominantly recognized in elderly people and repeatedly causes a huge subcortical hemorrhage. Some cases of CAA can cause secondary subarachnoid hemorrhage (SAH), but cases in which it causes primary SAH is very rare. We describe a valuable reference case of a 75-year-old man in whom a restricted SAH recurred in the cortical sulcus following a huge subcortical hemorrhage. He presented with an unknown restricted SAH in the left frontal sulcus twice before neck clipping for a right IC-PC unruptured aneurysm. Postoperative computed tomography (CT) revealed a recurrent SAH in the left frontal lobe, and it spread gradually. T2(*) weighted imaging (T2(*)WI) revealed subarachnoid hemosiderosis and superficial cortical hemosiderosis in the frontal and parietal lobe. On the 21st postoperative day, he suddenly presented right hemiplegia and a huge subcortical hemorrhage was observed in the left frontal lobe on CT. Emergent removal of the hematoma was performed, but the patient had become bedridden. Pathological diagnosis of CAA was made. A recurrent restricted SAH in the frontal sulcus might have been a warning sign of a huge subcortical hemorrhage. In the aging society, a radiological prediction of CAA is very important. Although it is generally thought to be very difficult, T2(*)WI may be useful for predicting CAA. When we plan surgery for elderly people, we must always take CAA into consideration.