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Yamahachi, H.

Publications and source records attributed to Yamahachi, H..

6 recordsLinked to original sources
bioRxiv2025

The alternative initiation factor eIF2A regulates 40S subunit turnover in ribosome-associated quality control

The noncanonical translation initiation factor eIF2A plays critical roles in diverse cellular processes, including the integrated stress response, neurodegeneration and tumorigenesis. However, the precise molecular mechanism underlying eIF2As function remains poorly understood. Here, we exploit a TurboID-based proximity labeling combined with mass spectrometry to systematically map the interactome of eIF2A during homeostasis and stress. Combining polysome gradients with TurboID, we zoom into the interactions of eIF2A with the 40S small ribosomal subunit and map the eIF2A binding site close to the mRNA entry channel. We identify a network of interactors that link eIF2A to ribosome-associated quality control, including its strong interaction with G3BP1-USP10 complexes as well as RPS2 and RPS3. In the absence of eIF2A, RPS2 and RPS3 ubiquitination is diminished specifically upon ribosome stalling. 40S-specific footprinting in eIF2A knockout cells shows minimal changes in 5UTR occupancy, consistent with a limited role for eIF2A in translation initiation. Using dynamic SILAC mass spectrometry, we characterize the novel function of eIF2A in ribosome-associated quality control and show that eIF2A antagonizes USP10-dependent rescue of 40S ribosomes, resulting in altered turnover of 40S subunits upon cellular stress. Collectively, our study identifies a previously unknown link between eIF2A and ribosome-associated quality control, implies that eIF2A promotes translation fidelity by tuning 40S ribosome rescue under stress and warrants further investigations into the role of ribosome-associated quality control in tumorigenesis.

molecular biology↗
bioRxiv2025

In vivo single-cell CRISPR screening for microproteins identifies a critical ribosomal component

The dark proteome includes a rapidly expanding catalog of microproteins with unknown functions that have been historically ignored in genome annotations. Here, we exploit an in vivo single-cell CRISPR screening strategy in the mouse epidermis to systematically investigate the tissue-wide function of microproteins. We document the global and cell-type-specific roles of microproteins during epidermal development and homeostasis at single-cell transcriptomic resolution. Focusing on select candidates, we identify a novel microprotein on Gm10076, identical to the ribosomal intersubunit bridge protein RPL41, whose perturbation strongly impairs proliferation and protein synthesis. Employing ribosome profiling and RNA sequencing, we show that Gm10076 perturbation profoundly reshapes the translational landscape. Contrary to its prior classification as nonessential, we find that the ribosomal protein RPL41 is essential for cellular proliferation, warranting further investigation into its role as an intersubunit bridge in the translational machinery. Together, our study comprehensively charts the tissue-wide functional landscape of the dark proteome, uncovers a second Rpl41 gene critical for ribosome function and establishes a basis for exploring the impact of microproteins on disease pathogenesis.

genetics↗
bioRxiv2024

In vivo single-cell ribosome profiling reveals cell-type-specific translational programs during aging

Somatic stem cells are characterized by their low overall protein synthesis rates, a feature implicated in driving their stemness. However, how aging reshapes the translational landscape of stem cells and how these changes impact their regenerative capacity remains poorly understood. Here, we present an in vivo single-cell ribosome profiling strategy to monitor tissue-wide translational landscapes of the young and aged mouse epidermis. By implementing ribosomal elongation-inhibited cell isolation and switching to RNase I ribonuclease for generating ribosomal footprints, we expand the applicability of single-cell ribosome profiling to in vivo systems and facilitate the evaluation of triplet periodicity, a hallmark of high-quality ribosome profiling data. Leveraging this strategy and integrating ribosome profiling with single-cell RNA sequencing, we document the in vivo translational landscapes of the major epidermal cell types, outline cell-type-specific translational efficiencies and capture heterogeneity in differentiation commitment within stem cell populations. Notably, we identify a pronounced translational reprogramming of AP-1 subunits specifically in aged epidermal stem cells, with functional consequences for keratinocyte behavior. Our study illustrates the power of in vivo single-cell ribosome profiling to map cell-type-specific translational programs and offers a scalable strategy for tissue-wide interrogation of translational landscapes at single-cell resolution.

genomics↗
bioRxiv2021

Monitoring the 5`UTR landscape reveals 5`terminal oligopyrimidine (TOP) motif switches to drive translational efficiencies

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here we use long-read sequencing to document the 5and 3untranslated region (UTR) isoform landscape of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. These 5UTR isoforms with identical coding sequences either include or exclude 5terminal oligopyrimidine (TOP) motifs and result in vastly altered translational efficiencies of the mRNA. Our findings suggest that switching between TOP and non-TOP motif-containing 5UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5UTR sequence.

cancer biology↗
bioRxiv2017

Welfare of zebra finches used in research

Over the past 50 years, songbirds have become a valuable model organism for scientists studying vocal communication from its behavioral, hormonal, neuronal, and genetic perspectives. Many advances in our understanding of vocal learning result from research using the zebra finch, a close-ended vocal learner. We review some of the manipulations used in zebra finch research, such as isolate housing, transient/irreversible impairment of hearing/vocal organs, implantation of small devices for chronic electrophysiology, head fixation for imaging, aversive song conditioning using sound playback, and mounting of miniature backpacks for behavioral monitoring. We highlight the use of these manipulations in scientific research, and estimate their impact on animal welfare, based on the literature and on data from our past and ongoing work. The assessment of harm-benefits tradeoffs is a legal prerequisite for animal research in Switzerland. We conclude that a diverse set of known stressors reliably lead to suppressed singing rate, and that by contraposition, increased singing rate may be a useful indicator of welfare. We hope that our study can contribute to answering some of the most burning questions about zebra finch welfare in research on vocal behaviors.

animal behavior and cognition↗