Biology subjects

Imataka, H.

Publications and source records attributed to Imataka, H..

4 recordsLinked to original sources

bioRxiv2025

Canonical translation factors eIF1A and eIF5B modulate the initiation step of repeat-associated non-AUG translation.

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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/654993v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@160e2adorg.highwire.dtl.DTLVardef@1c1cc8forg.highwire.dtl.DTLVardef@5d01d0org.highwire.dtl.DTLVardef@220507_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Reconstitution of C9orf72 (GGGGCC) repeat-associated non-AUG translation with purified components.

Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is 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 G4C2 repeat. 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 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. Significance statementEvery 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.

biochemistry↗

bioRxiv2022

Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes

Robust translation elongation of any given amino acid sequence is a prerequisite to shape proteomes. Nevertheless, nascent peptides could destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains stochastically can induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in the N-terminal regions could prematurely terminate translation, producing premature products as peptidyl-tRNA species. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we found 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. Indeed, a bioinformatics analysis revealed that the N-terminal regions of ORFs avoid D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

biochemistry↗

bioRxiv2021

Distinct roles and actions of PDI family enzymes in catalysis of nascent-chain disulfide formation

The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains (RNC) of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with short segments exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Similarly to ERp46, a PDI mutant with an occluded substrate-binding pocket displayed shorter-time RNC binding and higher efficiency in disulfide introduction than wild-type PDI. Altogether, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation in RNC when longer nascent chains emerge out from ribosome.

biochemistry↗