Browsing by keyword "Yeast"

Now showing items 1-2 of 2

    • Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo
      Zeidan, Quira; He, Feng; Zhang, Fan; Zhang, Hongen; Jacobson, Allan; Hinnebusch, Alan G. (2018-12-07)
      Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Delta mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Delta dhh1Delta double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Delta mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Delta and dhh1Delta mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

    • Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae
      Mangkalaphiban, Kotchaphorn; He, Feng; Ganesan, Robin; Wu, Chan; Baker, Richard E.; Jacobson, Allan (2021-04-20)
      Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3'-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5' of the stop codon, six nucleotides 3' of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3'-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3'-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3'-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.