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Yoshihisa, T.

Publications and source records attributed to Yoshihisa, T..

2 recordsLinked to original sources
bioRxiv2020

Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

eIF2 phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

molecular biology↗
bioRxiv2020

OTTER, a new method quantifying absolute amounts of tRNAs

To maintain optimal proteome, both codon choice of each mRNA and supply of aminoacyl-tRNAs are two principal factors in translation. Recent reports revealed that tRNAs are more dynamic in amount than we had expected. High-throughput methods such as RNA-Seq or microarray are versatile for comprehensive analyses of changes in individual tRNA amounts, but they suffer from lack of assessment of signal production efficiency of individual tRNA species. Thus, they are not the perfect choice to measure absolute amounts of tRNA. Here, we introduce a novel method for this purpose, oligonucleotide-directed three-primer terminal extension of RNA (OTTER), which employs fluorescence labeling at the 3-terminus of a specific tRNA by optimized reverse primer extension and an assessment step of each labeling efficiency by Northern blotting. We quantified absolute amounts of 34 individual and 4 pairs of isoacceptor tRNAs out of the total 42 nuclear-encoded isoacceptors in the yeast Saccharomyces cerevisiae. We revealed that the amounts of tRNAs are in the range of 0.030-0.73 pmol/g RNA in the yeast cells logarithmically grown in a rich glucose medium. The tRNA amounts seem to be regulated at the isoacceptor level by a few folds according to physiological growing conditions. Data obtained by OTTER are poorly correlated with those by simple RNA-Seq and only marginally with those by microarray. However, the OTTER data are good agreement with those by 2D-gel analysis of in vivo radiolabeled RNA samples. Thus, OTTER is a suitable method for quantifying absolute amounts of tRNAs in the isoacceptor resolution.

molecular biology↗