Abstract
Transfer RNA (tRNA) from all domains of life contains multiple modified nucleosides, the functions of which remain incompletely understood. Genetic interactions between tRNA modification genes in Saccharomyces cerevisiae suggest that different tRNA modifications collaborate to maintain translational efficiency. Here we characterize such collaborative functions in the ochre suppressor tRNA SUP4. We quantified ochre read-through efficiency in mutants lacking either of the 7 known modifications in the extended anticodon stem loop (G26-C48). Absence of U34, U35, A37, U47 and C48 modifications partially impaired SUP4 function. We systematically combined modification defects and scored additive or synergistic negative effects on SUP4 performance. Our data reveal different degrees of functional redundancy between specific modifications, the strongest of which was demonstrated for those occurring at positions U34 and A37. SUP4 activity in the absence of critical modifications, however, can be rescued in a gene dosage dependent fashion by TEF1 which encodes elongation factor eEF1A required for tRNA delivery to the ribosome. Strikingly, the rescue ability of higher-than-normal eEF1A levels extends to tRNA modification defects in natural non-suppressor tRNAs suggesting that elevated eEF1A abundance can partially compensate for functional defects induced by loss of tRNA modifications.
Highlights
Transfer RNA is known for the presence of extensive post-transcriptional modifications
These modifications are introduced by well characterized modifiers that can be inactivated by single gene deletions[10,37,38,39,40,41]
The presence of SUP4 entirely suppressed red pigmentation in the wild type and loss of ELP3, MOD5 (i6A) and PUS7 (ψ35) partially restored pigment formation, albeit to a lesser extent compared to the parental strain lacking SUP4 Transfer RNA (tRNA) altogether (Fig. 1B)
Summary
Transfer RNA (tRNA) is known for the presence of extensive post-transcriptional modifications. Some of the non-standard ribonucleosides are thought to be important for tRNA stability and folding or to improve codon-anticodon recognition[1,2,3]. The latter has been attributed to the wobble uridine modification. 5-methoxy-carbonyl-methyl-2-thiouridine (mcm5s2U)[4,5,6,7] In yeast, this modification is naturally present in tRNALysUUU, tRNAGlnUUG and tRNAGluUUC and is formed by two separate pathways which mediate mcm[5] side chain addition at position 5 of the uracil base and exchange of the oxygen for sulfur at position 2 (wobble uridine thiolation)[8]. Modifications at distinct positions of the anticodon loop might cooperatively promote tRNA function, an idea that is further supported by in vitro studies indicating an anticodon-prestructuring role shared between U34 and
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