今高 寛晃

The genomes of various RNA viruses and a subset of human genes contain structured RNA elements termed internal ribosomal entry sites (IRESs) to initiate translation in a cap-independent manner. The well-studied IRES from Hepatitis C virus (HCV) binds to eukaryotic initiation factor 3 (eIF3), but how the HCV IRES harnesses eIF3 for viral translation remains unclear. Here, we determined multiple cryo-EM structures in which the HCV IRES binds simultaneously to the ribosome and eIF3, covering steps from initiation to elongation. The eIF3 core subunits are displaced from the ribosome by binding more tightly to subdomain IIIb of the HCV IRES. However, cross-linking mass spectrometry suggested that the eIF3 noncore subunits in the HCV-IRES-mediated elongation complex remain in similar positions on the ribosome to those observed in the cap-mediated initiation complex. This currently determined configuration of eIF3 core and noncore subunits reveals the mechanisms through which the HCV IRES overcomes the competition with the host mRNA and promotes viral mRNA translation by utilizing eIF3. Interestingly, cryo-EM structures also revealed that the N-terminal domain of the eIF3 c-subunit (eIF3c-NTD) binds to the large ribosomal subunit (60S) during elongation. These findings suggest that eIF3 contributes to HCV IRES-mediated translation not only during initiation but also elongation and potentially in reinitiation. The interaction between the eIF3c-NTD and the 60S ribosome is likely to occur in general translation processes as well, contributing to 60S joining or eIF3 stabilization on the elongating ribosome.

Although eukaryotic initiation factor 2D (eIF2D) is implicated in translation initiation, reinitiation, and ribosome recycling, its precise role remains unclear. Here, we show that eIF2D promotes 40S ribosome recycling during intrinsic ribosome destabilization (IRD), a process in which ribosomes stochastically destabilize while translating proteins with consecutive acidic amino acids at their NH2-terminus. Unrecycled 40S ribosomes accumulate in eIF2D-deficient cells, leading to 80S ribosome stalling. Selective translation complex profiling (TCP-seq) reveals that eIF2D preferentially associates with IRD-prone regions. The winged helix domain, unique to eIF2D but absent in MCTS1-DENR, enhances its binding to 40S subunits, but likely clashes with ABCE1 during stop-codon-associated recycling. Loss of eIF2D reduces the expression of IRD-inducing proteins, including splicing factors. Together, these findings define a previously unappreciated role for eIF2D in 40S recycling and clarify its mechanistic divergence from the MCTS1-DENR complex.

Nucleotide repeat expansions, such as the GGGGCC repeats in C9orf72, associated with C9-ALS, are linked to neurodegenerative diseases. These repeat sequences undergo a non-canonical translation known as repeat-associated non-AUG (RAN) translation. Unlike canonical translation, RAN translation initiates from non-AUG codons and occurs in all reading frames. To identify potential regulators of RAN translation, we employed a bottom-up approach using a human factor-based reconstituted cell-free translation system to recapitulate RAN translation. This approach revealed that omission of either eIF1A or eIF5B enhanced the translation in all reading frames of C9orf72-mediated RAN translation (C9-RAN), suggesting that eIF1A and eIF5B act as repressors of RAN translation. eIF1A and eIF5B are known to contribute to the fidelity of translation initiation. In HEK293T cells, double knockdown of eIF1A and eIF5B further promoted C9-RAN compared to single knockdowns, indicating that these factors regulate C9-RAN through distinct initiation steps. Furthermore, under eIF1A knockdown conditions, the enhancement of RAN translation via the integrated stress response (ISR) was not observed in HEK293T cells, indicating that eIF1A is involved in the ISR-mediated non-AUG translation.

Programmed-1 ribosomal frameshifting (-1 PRF) is a translational mechanism adopted by some viruses, including SARS-CoV-2. To find a compound that can inhibit -1 PRF in SARS-CoV-2, we set up a high-throughput screening system using a HeLa cell extract-derived cell-free protein synthesis (CFPS) system. A total of 32,000 compounds were individually incubated with the CFPS system programmed with a -1 PRF-EGFP template. Several compounds were observed to decrease the -1 PRF-driven fluorescence, and one of them had some suppressive effect on -1 PRF of a SARS-CoV-2 genome sequence in transfected cells. Thus the CFPS system can be used as a tool for a high-throughput screening of chemicals.

Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5' cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.

OBJECTIVES: Recent studies demonstrate that extracellular-released aminoacyl-tRNA synthetases (aaRSs) play unique roles in immune responses and diseases. This study aimed to understand the role of extracellular aaRSs in the pathogenesis of rheumatoid arthritis (RA). METHODS: Primary macrophages and fibroblast-like synoviocytes were cultured with aaRSs. aaRS-induced cytokine production including IL-6 and TNF-α was detected by ELISA. Transcriptomic features of aaRS-stimulated macrophages were examined using RNA-sequencing. Serum and synovial fluid (SF) aaRS levels in patients with RA were assessed using ELISA. Peptidyl arginine deiminase (PAD) 4 release from macrophages stimulated with aaRSs was detected by ELISA. Citrullination of aaRSs by themselves was examined by immunoprecipitation and western blotting. Furthermore, aaRS inhibitory peptides were used for inhibition of arthritis in two mouse RA models, collagen-induced arthritis and collagen antibody-induced arthritis. RESULTS: All 20 aaRSs functioned as alarmin; they induced pro-inflammatory cytokines through the CD14-MD2-TLR4 axis. Stimulation of macrophages with aaRSs displayed persistent innate inflammatory responses. Serum and SF levels of many aaRSs increased in patients with RA compared with control subjects. Furthermore, aaRSs released PAD4 from living macrophages, leading to their citrullination. We demonstrate that aaRS inhibitory peptides suppress cytokine production and PAD4 release by aaRSs and alleviate arthritic symptoms in a mouse RA model. CONCLUSIONS: Our findings uncovered the significant role of aaRSs as a novel alarmin in RA pathogenesis, indicating that their blocking agents are potent antirheumatic drugs.

Abstract Nucleotide repeat expansion of GGGGCC (G₄C₂) in the non-coding region ofC9orf72is the most common genetic cause underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G₄C₂-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G₄C₂repeat. Additionally, we observed ribosomal frameshifting from the poly Gly-Ala dipeptide frame to other frames during the elongation process. Furthermore, the initiation mechanism proceeded in a 5’ cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G₄C₂repeats enhanced translation, we discovered that the expansion of the G₄C₂repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism. Significance statement Every cellular protein undergoes synthesis through a process known as translation. While the fundamental aspects of translation have been established, recent advancements have unveiled various noncanonical translation pathways, including the translation originating from “noncoding” RNAs. Within this context, certain neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), are linked to the translation of noncoding RNAs, referred to as repeat-associated non-AUG (RAN) translation, the underlying mechanism of which remains controversial. To dissect the complicated nature of RAN translation, this study employs a reconstituted cell-free translation system comprised of human translation factors. By reconstituting RAN translation utilizing a minimal set of factors, this bottom-up approach not only facilitates the elucidation of its mechanism but also offers a distinctive avenue for pharmaceutical development.

Robust translation elongation of any given amino acid sequence is required to shape proteomes. Nevertheless, nascent peptides occasionally destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains can stochastically induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, using budding yeast and a human factor-based reconstituted translation system, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in their N-terminal regions alter canonical ribosome dynamics, stochastically aborting translation. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we find many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk of failure. Indeed, a bioinformatics analysis reveals that the N-terminal regions of ORFs lack D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

In vitro reconstitution of whole cellular events is one of the important goals in synthetic biology. Using a cell-free protein synthesis (CFPS) system reconstituted with human translation factors and chaperones, we reproduced the biogenesis of β-actin, synthesis, folding, and polymerization in a test tube. This system enabled us to define which step of the β-actin biogenesis was defective in genetic mutations related to diseases. Hence, the CFPS system reconstituted with human factors may be a useful tool for analyzing proteostasis in eukaryotes.

Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3' untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3' UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3' UTRs. The 3' UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3' UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3' UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3' UTRs of mRNAs may encode various products.

<title>Abstract</title> Antizyme (AZ) interacts with ornithine decarboxylase, which catalyzes the first step of polyamine biosynthesis and recruits it to the proteasome for degradation. Synthesizing the functional AZ protein requires transition of the reading frame at the termination codon. This programmed +1 ribosomal frameshifting is induced by polyamines, but the molecular mechanism is still unknown. In this study, we explored the mechanism of polyamine-dependent +1 frameshifting using a human cell-free translation system. Unexpectedly, spermidine induced +1 frameshifting in the mutants replacing the termination codon at the shift site with a sense codon. Truncation experiments showed that +1 frameshifting occurred promiscuously in various positions of the AZ sequence. The probability of this sequence-independent +1 frameshifting increased in proportion to the length of the open reading frame. Furthermore, the +1 frameshifting was induced in some sequences other than the AZ gene in a polyamine-dependent manner. These findings suggest that polyamines have the potential to shift the reading frame in the +1 direction in any sequence. Finally, we showed that the probability of the sequence-independent +1 frameshifting by polyamines is likely inversely correlated with translation efficiency. Based on these results, we propose a model of the molecular mechanism for AZ +1 frameshifting.

<title>Abstract</title> We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

<title>Abstract</title> We developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors and programmed by CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing the active reporter protein, nanoLuciferase, with a molecular weight of 19 kDa. The protein synthesis by the system is appropriately regulated by controlling its composition, including translation factors, amino acids and antibiotics. We found that a high eEF1A concentration relative to the ribosome concentration is critically required for efficient IRES-mediated translation initiation, to ensure its dominance over IRES-independent random internal translation initiation.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.