Yeast applied readthrough inducing system (YARIS): an invivo assay for the comprehensive study of translational readthrough

Abstract

Stop codon readthrough—the decoding of a stop codon by a near-cognate tRNA—is employed by viruses to balance levels of enzymatic and structural proteins and by eukaryotic cells to enable isoform-specific protein synthesis in response to external stimuli. Owing to the prevalence of premature termination codons in human disease, readthrough has emerged as an attractive therapeutic target. A growing list of various features, for example the +4 nucleotide immediately following the stop codon, modulate readthrough levels, underscoring the need for systematic investigation of readthrough. Here, we identified and described a complete group of yeast tRNAs that induce readthrough in the stop-codon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs). These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporter-based assay enabling simultaneous detection of readthrough levels at all twelve stop-codon tetranucleotides and as a function of the complete set of rti-tRNAs. We demonstrate the utility of YARIS for systematic study of translation readthrough by employing it to interrogate the effects of natural rti-tRNA modifications, as well as various readthrough-inducing drugs (RTIDs). This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.

Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

“…While translation termination is generally the predominant reaction at stop codons, termination efficiencies do vary considerably between different stop codon contexts. Many factors have been reported to influence the probability of termination, readthrough, or frameshifting (where the ribosome slides on an mRNA, changing the frame of translation) including the identity of the stop codon and surrounding sequence contexts (Anzalone, Zairis, Lin, Rabadan, & Cornish, 2019;Bonetti, Fu, Moon, & Bedwell, 1995;Floquet et al, 2012;Harrell et al, 2002;McCaughan, Brown, Dalphin, Berry, & Tate, 1995;Namy et al, 2001), proximal RNA structures (Firth, Wills, Gesteland, & Atkins, 2011;Steneberg & Samakovlis, 2001), RNA modifications (Karijolich & Yu, 2011), presence of RNA binding proteins (Amrani et al, 2004), and availability of aminoacylated nc-tRNA (Beznosková, Pavlíková, Zeman, Echeverría Aitken, & Valášek, 2019;Blanchet et al, 2015;Roy, Leszyk, Mangus, & Jacobson, 2015). Intriguingly, high rates of SCR have been documented in diverse organisms including viruses (Li & Rice, 1993;Wills, Gesteland, & Atkins, 1991), yeast (Namy et al, 2003;Williams, Richardson, Starkey, & Stansfield, 2004), flies (Dunn, Foo, Belletier, Gavis, & Weissman, 2013), and humans (Loughran et al, 2014(Loughran et al, , 2018.…”

Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

“…including the identity of the stop codon and surrounding sequence contexts (Anzalone, Zairis, Lin, Rabadan, & Cornish, 2019;Bonetti, Fu, Moon, & Bedwell, 1995;Floquet et al, 2012;Harrell et al, 2002;McCaughan, Brown, Dalphin, Berry, & Tate, 1995;Namy et al, 2001), proximal RNA structures (Firth, Wills, Gesteland, & Atkins, 2011;Steneberg & Samakovlis, 2001), RNA modifications (Karijolich & Yu, 2011), presence of RNA binding proteins (Amrani et al, 2004), and availability of aminoacylated nc-tRNA (Beznosková, Pavlíková, Zeman, Echeverría Aitken, & Valášek, 2019;Blanchet et al, 2015;Roy, Leszyk, Mangus, & Jacobson, 2015). Intriguingly, high rates of SCR have been documented in diverse organisms including viruses (Li & Rice, 1993;Wills, Gesteland, & Atkins, 1991), yeast (Namy et al, 2003;Williams, Richardson, Starkey, & Stansfield, 2004), flies (Dunn, Foo, Belletier, Gavis, & Weissman, 2013), and humans (Loughran et al, 2014.…”

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

“…We have recently published a comprehensive analysis of all natural tRNAs that are near-cognate with some of three stop codons in yeast in order to identify all those that are capable of efficient decoding of stop codons ( 47 , 48 ). This analysis uncovered altogether four so-called readthrough-inducing (rti) tRNAs - Trp, Cys, Gln and Tyr.…”

“…With respect to the latter, we recently demonstrated that it is the identity of the stop codon tetranucleotide that largely determines preferences for specific near-cognate tRNAs in such a way that new decoding rules could have been proposed, at least for the yeast genome ( 42 , 47–49 ). In particular, four highly readthrough-efficient near-cognate tRNAs (tRNA Trp , tRNA Cys , tRNA Gln , tRNA Tyr ) were identified with a defined stop codon tetranucleotide-specificity that we designated as readthrough-inducing tRNAs (rti-tRNAs) ( 47 , 48 ). Whether the same stop codon tetranucleotide decoding rules apply also on mammals remains to be determined.…”