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.

Figure 1.

tRNA^Tyr[tY(GUA)J2] and tRNA^Gln[tQ(CUG)M] are the specific readthrough substrates for the UAA-N and UAG-N tetranucleotides in budding yeast. (A) Schematics of Tyr and Gln tRNAs base-pairing with the indicated stop codon tetranucleotides. Only the nucleotides of the anticodon loop are shown with the third stop codon base N₃₄ (in gray) and N₃₂ indicated. (B) Increased gene dosage of tY(GUA)J2 or tQ(CUG)M tRNAs increases their cellular levels in vivo. Total RNAs were extracted from the PBH156 strain bearing a plasmid indicated at the top of each panel and 1 or 0.5 μg aliquots were loaded onto the Criterion Precast gels and subjected to northern blot with ³²P-labeled probes shown on the left. (C) Increased gene dosage of tY(GUA)J2 elevates readthrough levels on all four UAA-N tetranucleotides. The PBH156 was transformed with either empty vector (EV) or high copy (hc) tY(GUA)J2 and the resulting transformants were grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs YEp-R/T-CAAC-L; YEp-R/T-UAAC-L; PBB157; PBB161 and PBB162, as described in ‘Materials and Methods’ section. Readthrough values are represented as mean ± SD from quintuplicates (n = 5) and each experiment was repeated at least three times. (D) The impact of an increased gene dosage of tY(GUA)J2 or tQ(CUG)M on the UAG-N readthrough. The PBH140 derivative was transformed with either EV, hc tY(GUA)J2 or hc tQ(CUG)M and the resulting transformants were grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs YEp-R/T-CAAC-L; YEp-R/T-UAGC-L; PBB159; PBB160 and PBB158, as described in ‘Materials and Methods’ section. Readthrough values are represented as mean ± SD from quintuplicates (n = 5) and each experiment was repeated at least three times.

Figure 2.

Expanded decoding rules defining readthrough inducing tRNAs (rti-tRNAs) from the pool of all near-cognate tRNAs. (A) A modified schematic of the genetic code to highlight all existing near-cognate tRNAs for all three stop codons; numbers of genes coding for individual near-cognate tRNAs occurring in the entire Saccharomyces cerevisiae genome are given (source SGD). (B) A schematic illustrating the expanded decoding rules for specific incorporation of individual rti-tRNAs at individual stop codons determined by the nucleotide immediately following the stop codon (+4; fourth letter).

Figure 3.

YARIS. (A) An experimental outline of YARIS. Yeast cells of interest are transformed with two plasmids; the first plasmid bears an rti-tRNA of interest or an EV, while the second one carries a dual luciferase readthrough reporter with either varying stop codon tetranucleotides or a control sense codon (CAG). Cells are grown under selected conditions and lysates are prepared and subjected to measurements of luciferase activity according to the vendor. Readthrough values are normalized to cells bearing the EV and the log₂ fold increases in readthrough are expressed as heatmaps created using R. (B) The Log2 fold increase heatmap of the readthrough induction in the PBH156 strain expressing various combinations of rti-tRNAs (columns) with stop codon tetranucleotides (rows).

Figure 4.

Dissection of the readthrough effects of distinct tRNA modifications using YARIS. The Log2 fold increase heatmap of the readthrough induction in strains individually deleted for genes encoding selected tRNA modifying enzymes expressing various combinations of rti-tRNAs (columns) with stop codon tetranucleotides (rows).

Figure 5.

YARIS reveals rti-tRNA:tetranucleotide specific effects of aminoglycosides. (A) Quantification of cellular levels of four rti-tRNAs individually overexpressed in the H2879 strain. The upper panel summarizes fold-increases of overexpressed rti-tRNAs relative to an EV after normalization to the cellular level of 5.8S rRNA; the middle and bottom panels show the corresponding northern blots. Total RNAs were extracted from the H2879 strain bearing a high copy plasmid indicated at the top of each panel and 1 or 0.5 μg aliquots were loaded onto the Criterion Precast gels and subjected to northern blot with ³²P-labeled probes shown on the right. Northern blots were quantified using the Quantity One program and the signals were first normalized to the control 5.8S rRNA. The resulting values obtained with cells bearing an EV (hc EV) were then set to 1.00 and those obtained with cells overexpressing individual rti-tRNAs were expressed relative to the hc EV control. Standard deviations from three individual experiments are given. (B) The Log2 fold increase heatmap of the readthrough induction in strains expressing various combinations of rti-tRNAs (columns) with stop codon tetranucleotides (rows) that were subjected to the indicated aminoglycoside treatments. In particular, the H2879 strain was transformed with either EV; hc tW(CCA)G1; hc tC(GCA)P1; hc tY(GUA)J2 or hc tQ(CUG)M, and the resulting transformants were grown in SD only (−), or supplemented for six hours with either G418 (G418; final concentration 50μg/ml); paromomycin (PARO; 200 μg/ml), or tobramycin (TOBRA; 400 μg/ml); and processed for stop codon readthrough measurements as described above. For each stop codon tetranucleotide (shown to the left) the readthrough values from untreated cells bearing the ‘EV’ were set to 1 (column 1).