Identification of yeast tRNA Um44 2′-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNASer species

A characteristic feature of tRNAs is the numerous modifications found throughout their sequences, which are highly conserved and often have important roles. Um44 is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and unique to tRNASer in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNASer Um44 2′-O-methyltransferase. Trm44 was identified by screening a yeast genomic library of affinity purified proteins for activity and verified by showing that a trm44-Δ strain lacks 2′-O-methyltransferase activity and has undetectable levels of Um44 in its tRNASer and by showing that Trm44 purified from Escherichia coli 2′-O-methylates U44 of tRNASer in vitro. Trm44 is conserved among metazoans and fungi, consistent with the conservation of Um44 in eukaryotic tRNAs, but surprisingly, Trm44 is not found in plants. Although trm44-Δ mutants have no detectable growth defect, TRM44 is required for survival at 33°C in a tan1-Δ mutant strain, which lacks ac4C12 in tRNASer and tRNALeu. At nonpermissive temperature, a trm44-Δ tan1-Δ mutant strain has reduced levels of tRNASer(CGA) and tRNASer(UGA), but not other tRNASer or tRNALeu species. The trm44-Δ tan1-Δ growth defect is suppressed by addition of multiple copies of tRNASer(CGA) and tRNASer(UGA), directly implicating these tRNASer species in this phenotype. The reduction of specific tRNASer species in a trm44-Δ tan1-Δ mutant underscores the importance of tRNA modifications in sustaining tRNA levels and further emphasizes that tRNAs undergo quality control

Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5′–3′ exonucleases Rat1 and Xrn1

Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD) pathway, since mature tRNAVal(AAC) lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and deacylated at 37°C in a trm8-Δ trm4-Δ strain, resulting in temperature-sensitive growth. We show here that components of this RTD pathway include the 5′–3′ exonucleases Rat1 and Xrn1, and Met22, which likely acts indirectly through Rat1 and Xrn1. Since deletion of MET22 or mutation of RAT1 and XRN1 prevent both degradation and deacylation of mature tRNAVal(AAC) in a trm8-Δ trm4-Δ strain and result in healthy growth at 37°C, hypomodified tRNAVal(AAC) is at least partially functional and structurally intact under these conditions. The integrity of multiple mature tRNA species is subject to surveillance by the RTD pathway, since mutations in this pathway also prevent degradation of at least three other mature tRNAs lacking other combinations of modifications. The RTD pathway is the first to be implicated in the turnover of mature RNA species from the class of stable RNAs. These results and the results of others demonstrate that tRNA, like mRNA, is subject to multiple quality control steps