Short communicationA map of 5-methylcytosine residues in Trypanosoma brucei tRNA revealed by sodium bisulfite sequencing

•The T. brucei tRNAs analyzed contain between 1 and 4 5-methylcytosines.•Cytosine residues near the variable region-TψC junction are frequently methylated.•T. brucei tRNAGly(GCC) displays cytosine methylation at C40 of the anticodon stem.•There is no evidence for 5-methylcytosine at C38 in the T. brucei anticodon loop.

In protozoan parasites, there is little information on the presence of covalent RNA modifications which comprise the epitranscriptome. Therefore, we determined if T. brucei tRNAAsp(GUC), tRNAGly(GCC), tRNAVal(AAC), and tRNATyr(GUA) contain 5-methylcytosines via RNA bisulfite sequencing. Most tRNAs examined have at least one 5-methylcytosine at the variable region-TψC junction. Only tRNAGly(GCC) displayed methylation of C40 in the anticodon stem, and there was partial methylation at this site. There is no evidence for methylation of C38 in the anticodon loop in the tRNAs analyzed. Analysis of tRNATyr(GUA) demonstrates that both unspliced and spliced molecules contain C48 methylation, indicating tRNA cytosine methylation can precede tRNA splicing. Overall, our data indicate that T. brucei tRNAs contain 5-methylcytosine residues in some, but potentially not all standard eukaryotic positions. The levels of cytosine methylation of different T. brucei tRNAs vary, suggesting the presence of a mechanism for methylation control.

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