Biology subjects

Tomuro, K.

Publications and source records attributed to Tomuro, K..

4 recordsLinked to original sources

bioRxiv2025

Sequence grammar and dynamics of subcellular translation revealed by APEX-Ribo-Seq

Local translation at specific subcellular regions is proposed to define appropriate protein destinations and functions. However, our understanding of local translation still remains far from complete due to a lack of versatile analytical tools. Here, we developed a new method, termed APEX-Ribo-Seq, that integrates ribosome profiling (Ribo-Seq) with APEX2-based proximity labeling. We mapped local translation across 14 distinct organelles and identified over 3,000 genes undergoing localized translation, encoding components of complexes that function at these specific sites. RNA language-model predictors captured compartment-associated information beyond protein sequence, and model-derived attribution nominated sequence-sensitive transcript regions for mechanistic follow-up. Gene-set-level RBP enrichment and knockdown experiments separately identified RBPs associated with compartment-specific translation. The utility of APEX-Ribo-Seq was demonstrated in primary neurons, revealing compartment-specific translatomes in dendrites, axons, and presynapses. Extending this approach in vivo, we applied APEX-Ribo-Seq to germ granules in Drosophila embryos, profiling the temporal transition from translationally silent mRNA storage to localized translation during early embryogenesis. Our results provide a comprehensive view of the spatiotemporal translatome and its regulation.

molecular biology↗

bioRxiv2023

Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster

N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) lost the elongated spermatids and mature sperm, which was fully rescued by a Mettl1-transgene expression, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO resulted in a loss of m7G modification on a subset of tRNAs and a decreased level of tRNA expression. Strikingly, overexpression of the translational elongation factor, EF11, which can compete with the rapid tRNA decay (RTD) pathway in S. cerevisiae, significantly counteracted the sterility of Mettl1-KO males, supporting a critical role of m7G modification of tRNAs in spermatogenesis. Ribosome profiling showed that Mettl1-KO led to the ribosome stalling at codons decoded by tRNAs that were reduced in expression. Mettl1-KO also significantly reduced the translation efficiency of genes involved in elongated spermatid formation and sperm stability. These findings reveal a developmental role for m7G tRNA modifications and indicate that m7G modification-dependent tRNA stability differs among tissues.

molecular biology↗

bioRxiv2023

Complexity and dynamics of in organello translation landscape assessed by high-resolution mitochondrial ribosome profiling

Since mitochondrial translation serves the essential subunits of the OXPHOS complex that produces ATP, exhaustive, quantitative, and high-resolution delineation of mitoribosome traversal is needed. Here, we developed a technique for high-resolution mitochondrial ribosome profiling and revealed the intricate regulation of mammals in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript and the elongation rate, initiation rate, and lifetime rounds of translation of individual transcripts. We also surveyed the impacts of modifications at the anticodon stem loop in mt-tRNAs, including all possible modifications at the 34th position, by deleting the corresponding enzymes and harnessing patient-derived cells. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed cryptic translation initiation sites at subcognate codons and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.

molecular biology↗

bioRxiv2023

Absolute calibration of ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to determine overall ribosome numbers in transcripts. Here, we overcame these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches measures the absolute number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method revealed the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy transforms ribosome profiling technique from relative to absolute quantification of translation.

molecular biology↗