Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution

3-Methylcytidine (m3C), a prevalent modification of tRNAs, was recently identified in eukaryotic mRNAs. However, its precise distribution and formation mechanisms in mRNAs remain elusive. Here we develop a novel approach, m3C immunoprecipitation and sequencing (m3C-IP-seq), utilizing antibody enrichment to profile the m3C methylome at single-nucleotide resolution. m3C-IP-seq captures 12 m3C modification sites in cytoplasmic tRNA isoacceptors and 2 in mitochondrial tRNA isoacceptors. Moreover, m3C-IP-seq permits the comprehensive profiling of m3C sites in mRNAs and lncRNAs, with their presence reliant on a nuclear isoform of METTL8. A significant proportion of m3C sites is concentrated in the 3' untranslated region (3' UTR) of mRNAs. Additionally, m3C methylation is dynamic and responds to hypoxia. Functionally, m3C formation in the 3' UTR is associated with mRNA degradation. Collectively, our data demonstrate the widespread presence of m3C modification in the human transcriptome and provide a resource for functional studies of m3C-mediated RNA metabolism. Overall design: [dataset 1] 6 samples. Duplicates for m3C-IP-seq of tRNA samples, including three sections of tRNA m3C Input, tRNA m3C IP, and tRNA m3C Dm in the HEK293T cell line. [dataset 2] 6 samples. Duplicates for m3C-IP-seq of polyA+ RNA samples, including three sections of m3C Input, m3C IP, and m3C Dm in the HEK293T cell line. [dataset 3] 5 samples. m3C-IP-seq of harboring various abundances of m3C spike-ins, including 0%, 20%, 40%, 80%, and 100% abundance in synethesisd m3C oligos. [dataset 4] 3 samples. m3C-IP-seq of tRNA samples mixed with 10% m3C spike-ins, including three sections of tRNA m3C Input, tRNA m3C IP, and tRNA m3C Dm in the HEK293T cell line. [dataset 5] 2 samples. RNA-seq of RNA samples from HEK293T cells with or without METTL8.