Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the ${\hbox{ pre-tRNA }}_{\left(\hbox{ CAA }\right)}^{\hbox{ Leu }}$

Abstract

We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast pre-tRNA(CAA)Leu. Using transcripts of intron-less and intron-containing human tRNA(CAA)Leu genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m5C34 formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNALeu transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m5C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of human pre-tRNA(CAA)Leu and identification of human gene encoding tRNA methylase responsible for this reaction.

The Mouse Cytosine-5 RNA Methyltransferase NSun2 Is a Component of the Chromatoid Body and Required for Testis Differentiation

“…NSun2 is the orthologue of Saccharomyces cerevisiae Trm4 tRNA methylase (47,59,60). NSun2 catalyzes the formation of cytosine-5 methylation (m 5 C) in several tRNAs in vivo in tissues, including skin, liver, and testis (3,21).…”

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

“…Here, a sequence pattern (C32, A37, C38, C40) was suggested to be required for target recognition by DNMT2 (Mueller et al 2013) and it was recently shown that a GG dinucleotide in the variable loop works as an anti-determinant for DNMT2 methylation (Shanmugam et al 2014). The formation of m 5 C34 in tRNA Leu-CAA by NSUN2 requires the presence of the intron in the pre-tRNA (Brzezicha et al 2006). In the case of NSUN6, the CCA tail contributes to target recognition but it is so far not known, which additional sequence or structural information of the tRNA is required for its recognition as an NSUN6 substrate.…”

Genetic Defects Underlie the Non-syndromic Autosomal Recessive Intellectual Disability (NS-ARID)

“…The NSUN2 (NCBI RefSeqGene: NG_028215.1) is composed of 19 exons and spans a 34 kb region on chromosome 5p15.31. The NSUN2 gene encodes a 767-amino acid protein of 86 kDa (UniProtKB # Q08J23) that shares 35% sequence identity with yeast ortholog Trm4 [70], and is expressed in the human brain at various times during development. Expression of an ortholog of NSUN2 was also detected in brain cells of mouse [71].…”