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

Abstract

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.