Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast

Abstract

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.

Keywords: GCN4; eRF1; programmed stop codon readthrough; termination; tetranucleotide; uORF.

FIGURE 1.

Cytosine immediately following any stop codon interferes with the eRF1 decoding in vivo. (A) Stop codon readthrough measured at all four UGA-N termination tetranucleotides in wt cells (SUP45) and two eRF1 mutants (sup45-M48I and sup45-Y410S). The 74D-694, L2327, and L2521 strains 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-UGAC-L; PBB75; PBB76; and PBB77, as described in Materials and Methods. Readthrough values are represented as mean ± SD from quintuplicates (n = 5) and each experiment was repeated at least three times. (B) Normalization of readthrough measurements from panel A; values measured for UGA-U of each of the four strains were set to one. (C) Same as in panel A, except that all four UAA-N termination tetranucleotides were examined. (D) Same as in panel A, except that all four UAG-N termination tetranucleotides were examined.

FIGURE 2.

The UGA-A tetranucleotide is preferentially read through by tryptophan nc-tRNA and the UGA-C, and UGA-G tetranucleotides are preferentially read through by cysteine nc-tRNA. (A) Schematics of Trp and Cys nc-tRNAs base-paring 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) Effect of the eIF3 presence in pre-TCs on UGA-N readthrough upon increased gene dosage of tryptophan nc-tRNA. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles were transformed with either empty vector (EV) or high copy (hc) tW(CCA)G1 and the resulting transformants were grown and processed for stop codon readthrough measurements as described in Figure 1. (C) Effect of the eIF3 presence in pre-TCs on UGA-N readthrough upon increased gene dosage of cysteine nc-tRNA. Essentially the same as in B, except that hc tC(GCA)P1 was used in place of hc tW(CCA)G1.

FIGURE 3.

Effect of the miscoding agent paromomycin on hc tryptophan or cysteine nc-tRNA incorporation at UGA-N termination tetranucleotides. (A) The PBH140 derivative bearing TIF35 wt was transformed with either empty vector (EV) or hc tW(CCA)G1, and the resulting transformants were grown in SD with 200 µg/mL paromomycin for 6 h and processed for stop codon readthrough measurements as described in Figure 1. (B) Essentially the same as in A, except that hc tC(GCA)P1 was used in place of hc tW(CCA)G1.

FIGURE 4.

Effect of the eRF1 (sup45-M48I) mutation with impaired decoding ability on hc tryptophan or cysteine nc-tRNA incorporation at UGA-N termination tetranucleotides. (A) The L2327 was transformed with either empty vector (EV) or hc tW(CCA)G1 and the resulting transformants were grown and processed for stop codon readthrough measurements as described in Figure 1. (B) Essentially the same as in A, except that hc tC(GCA)P1 was used in place of hc tW(CCA)G1.

FIGURE 5.

eIF3-promoted readthrough does not interfere with the eIF3-promoted REI on REI-permissive uORF1 from the GCN4 mRNA leader. (A) The 6-nt-long context immediately following stop codons of all four uORFs from the GCN4 mRNA leader allows eIF3-dependent readthrough with varying efficiency. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles were grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs pTH460; pTH477; PBB135; PBB136; PBB137; PBB138; and PBB139 as described in Materials and Methods. Changes in the measured readthrough values between TIF35 and tif35-KLF cells were analyzed by the Student's t-test (mean ± SD; n = 6) and shown to be statistically significant only for those cases marked with the asterisk (P < 0.01). (B) The 6-nt-long sequence preceding the stop codon of uORF1 nullifies the eIF3-mediated stimulation of stop codon readthrough on this REI-permissive uORF. The PBH140 derivative bearing the TIF35 wt allele was grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs pTH460; pTH477; PBB135; PBB136; PBB137; PBB138; PBB139; PBB140; PBB141; PBB142; PBB143; and PBB144 as described in Materials and Methods. Changes in the measured readthrough values between selected constructs were analyzed by the Student's t-test (mean ± SD; n = 6) and shown to be statistically significant only for those cases marked with the asterisk (P < 0.01).