Identification of yeast tRNA Um₄₄ 2′-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA^Ser 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 tRNA^Ser in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNA^Ser 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 tRNA^Ser and by showing that Trm44 purified from Escherichia coli 2′-O-methylates U₄₄ of tRNA^Ser 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 tRNA^Ser and tRNA^Leu. At nonpermissive temperature, a trm44-Δ tan1-Δ mutant strain has reduced levels of tRNA^Ser(CGA) and tRNA^Ser(UGA), but not other tRNA^Ser or tRNA^Leu species. The trm44-Δ tan1-Δ growth defect is suppressed by addition of multiple copies of tRNA^Ser(CGA) and tRNA^Ser(UGA), directly implicating these tRNA^Ser species in this phenotype. The reduction of specific tRNA^Ser 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.

Keywords

• S. cerevisiae
• tRNASer
• TAN1
• quality control