Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Abstract

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 and mRNA secondary structure in the 3′-UTR 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.

Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance

“…Sequence Alignment, Transcript Quantification, and Data Analyses. RNA-Seq and ribosome profiling reads were aligned to a yeast transcriptome (available at https://github.com/Jacobson-Lab/yeast_transcriptome_v5) using bowtie (Langmead et al, 2009) and transcript abundance were determined using RSEM (Li and Dewey, 2011), as described previously (Mangkalaphiban et al, 2021). For intron-containing genes, spliced ("mRNA") and unspliced ("pre-mRNA") isoforms were indexed as separate entries.…”

“…Therefore, reads arising from UPF factor transcripts were discarded from these and all subsequent ribosome profiling libraries analyzed. PCR duplicates identified via the 4N barcodes on either side of the read were removed (Mangkalaphiban et al, 2021). Read processing statistics for ribosome profiling libraries are provided in Table S1.…”

Systematic analysis of nonsense variants uncovers peptide release rate as a novel modifier of nonsense-mediated mRNA decay efficiency

“…Both these aspects are influenced by cis-acting elements (Baker & Hogg, 2017;Loughran et al, 2014;McCaughan et al, 1995;Pierson et al, 2016;Tork et al, 2004). For example, specific elements within the stop codon sequence context, as well as certain viral structures, can reduce termination fidelity (Baker & Hogg, 2017;Bonetti et al, 1995;Cridge et al, 2018;Loughran et al, 2014;Mangkalaphiban et al, 2021;Tork et al, 2004). Additionally, the identity of the nucleotide immediately after the stop codon and the peptide-tRNA hydrolysis rates of the amino acid preceding the stop codon impact the rate of peptide release (McCaughan et al, 1995;Pierson et al, 2016).…”

“…Both these aspects are influenced by cis-acting elements (14)(15)(16)(17)(18). For example, specific elements within the stop codon sequence context, as well as certain viral structures, can reduce termination fidelity (14)(15)(16)(19)(20)(21).…”

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArgand tRNALysisoacceptors in the P site

“…The key stop codon context determinant is the +4 position, i.e. the 3' nucleotide following the stop codon, which together with the stop codon itself forms the so-called the "extended stop codon" (Anzalone et al, 2019;Cridge et al, 2018;Harrell et al, 2002;Mangkalaphiban et al, 2024;Mangkalaphiban et al, 2021;McCaughan et al, 1995;Namy et al, 2001;Tate et al, 1995;Wangen & Green, 2020). The UGA stop codon followed by a 3' cytosine is the least efficient in promoting termination (Anzalone et al, 2019;Bonetti et al, 1995;Tate et al, 1996).…”