Crucial role of leaky initiation of uORF3 in the downregulation of HNT1 by ER stress.

eIF2α phosphorylation-mediated translational regulation is crucial for global repression of translation by various stresses, including the unfolded protein response (UPR) in eukaryotes. Although translational control during UPR has not been extensively investigated in S. cerevisiae, Hac1-mediated production of long transcripts containing uORFs was shown to repress the translation of histidine triad nucleotide-binding 1 (HNT1) mRNA. The present study showed that uORF3 is required for HNT1 expression, as well as down-regulating HNT1 translation. Translation initiation by uORF3 is inefficient, with uORF3 having a strong Kozak sequence efficiently repressing the translation of HNT1. We propose that leaky scanning of uORF3 is responsible for the downregulation of HNT1 during UPR.

Crucial role of ATP-bound Sse1 in Upf1-dependent degradation of the truncated product.

Up-frameshift (Upf) complex facilitates the degradation of aberrant mRNAs containing a premature termination codon (PTC) and its products in yeast. Here we report that Sse1, a member of the Hsp110 family, and Hsp70 play a crucial role in Upf-dependent degradation of the truncated FLAG-Pgk1-300 protein derived from PGK1 mRNA harboring a PTC at codon position 300. Sse1 was required for Upf-dependent rapid degradation of the FLAG-Pgk1-300. FLAG-Pgk1-300 was significantly destabilized in ATP hydrolysis defective sse1-1 mutant cells than in wild type cells. Consistently, Sse1 and Hsp70 reduced the level of an insoluble form of FLAG-Pgk1-300. We propose that the Sse1/Hsp70 complex maintains the solubility of FLAG-Pgk1-300, thereby stimulating its Upf-dependent degradation by the proteasomes.

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

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 (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.

The Ccr4-Not complex monitors the translating ribosome for codon optimality.

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.