m3C HAC-Seq analysis in HEK293T WT cells, METTL2A/2B knockout cells, METTL6 knockout cells, METTL2A/2B/6 knockout cells.

The 'epitranscriptome' includes a diversity of RNA modifications that influence gene expression. N3-methylcytidine (m3C) mainly occurs in the anticodon loop (position C32) of certain tRNAs yet its role is poorly understood. Here, using HAC-Seq, we report comprehensive METTL2A/B-, METTL6-, and METTL2A/B/6-dependent m3C profiles in human cells. METTL2A/B modifies most tRNA-Arginine and tRNA-Threonine members, whereas METTL6 modifies the tRNA-Serine family. However, decreased m3C32 on tRNA-Ser-GCT isodecoders was only observed with combined METTL2A/B/6 deletion. Ribo-Seq revealed altered translation of genes related to cell cycle and DNA repair pathways in METTL2A/2B/6-deficient cells, and these mRNAs are enriched in AGU codons that require tRNA-Ser-GCT for translation. These results, supported by reporter assays, help explain the observed slowed proliferation, altered cell cycle, and increased Cisplatin-sensitivity phenotypes of METTL2A/B/6-deficient cells. Thus, we define METTL2A/B/6-dependent methylomes and uncover a particular requirement of m3C tRNA modification for Serine codon-biased mRNA translation of cell cycle, and DNA repair genes. Overall design: Examination of m3C modification in HEK293T WT cells, METTL2A/2B knockout cells, METTL6 knockout cells, METTL2A/2B/6 knockout cells.