Tohru Yoshihisa

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3′-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p’s direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ₁₀ deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.

Saccharomyces cerevisiae has six synonymous tRNATrpCCA genes encoding the identical sequence, including their intronic region. They are supposed to express tRNATrpCCA in the same quality and quantity. Here, we generated single to quintuple deletion strains with all the possible combinations of the synonymous tRNATrpCCA genes to analyze whether those individual genes equally contribute cell viability and tRNA production. The quintuple deletion strains that only harbor tW(CCA)J, tW(CCA)M, or tW(CCA)P were viable but almost lethal while the other quintuple deletions showed moderately impaired growth. Theses growth differences were not obvious among the quadruple deletion strains, which expressed almost one third of mature tRNATrpCCA in the wild type. Therefore, no dosage compensation operates for tRNATrpCCA amount, and growth variations among the quintuple deletion strains may not simply reflect differences in tRNATrpCCA shortage. Yeast may retain the redundancy of tRNATrpCCA genes for a noncanonical function(s) beyond supply of the tRNA to translation.

ABSTRACT Yeasts generally grow with a highly glycolytic metabolism and restrain mitochondrial biogenesis except for some Fe/S proteins. Respiratory mitochondrial functions and biosynthesis pathways are well studied, however how cells coordinate basal fermentative mitochondrial functions is not fully understood. We show that a part of nuclear-encoded mitochondrial mRNAs, which preferentially translated in monosomes, are regulated by Puf3p upon glucose-rich media. Especially, of those monosome-enriched nuclear-encoded mitochondrial mRNAs, CAT5/COQ7 mRNA has a variant of the canonical Puf3p binding site on its 3’-UTR. Western blot analysis showed that Puf3p represses the translation of Cat5p regardless of fermentable or respiratory media. In vitro binding assay revealed that Puf3p directly binds to CAT5 mRNA via the non-canonical Puf3p binding site. Mutants harboring the substitution of the non-canonical Puf3p binding site in CAT5 mRNA grew normally but impaired Cat5p expressions apparently, indicating CAT5 mRNA is a bona fide Puf3p target. Overall, Puf3p, a general key modulator for nuclear-encoded mitochondrial mRNAs, fine-tunes translation of a subset of nuclear-encoded mitochondrial mRNAs including mRNAs with non-canonical Puf3p binding sites under the fermentation. This may be required to keeping the fundamental functions of yeast mitochondria at proper levels.

To maintain optimal proteome, both codon choice of each mRNA and supply of aminoacyl-tRNAs are two principal factors in translation. Recent reports have revealed that the amounts of tRNAs in cells are more dynamic than we had expected. High-throughput methods such as RNA-Seq and microarrays are versatile for comprehensive detection of changes in individual tRNA amounts, but they suffer from inability to assess signal production efficiencies of individual tRNA species. Thus, they are not the perfect choice to measure absolute amounts of tRNAs. Here, we introduce a novel method for this purpose, termed Oligonucleotide-directed Three-prime Terminal Extension of RNA (OTTER), which employs fluorescence-labeling at the 3'-terminus of a tRNA by optimized reverse primer extension and an assessment step of each labeling efficiency by northern blotting. Using this method, we quantified the absolute amounts of the 34 individual and 4 pairs of isoacceptor tRNAs out of the total 42 nuclear-encoded isoacceptors in the yeast Saccharomyces cerevisiae. We found that the amounts of tRNAs in log phase yeast cells grown in a rich glucose medium range from 0.030 to 0.73 pmol/µg RNA. The tRNA amounts seem to be altered at the isoacceptor level by a few folds in response to physiological growing conditions. The data obtained by OTTER are poorly correlated with those by simple RNA-Seq, marginally with those by microarrays and by microscale thermophoresis. However, the OTTER data showed good agreement with the data obtained by 2D-gel analysis of in vivo radiolabeled RNAs. Thus, OTTER is a suitable method for quantifying absolute amounts of tRNAs at the level of isoacceptor resolution.

<title>Abstract</title>eIF2α 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 (<italic>HAC1i</italic>) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (<italic>HNT1</italic>) 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.