Nucleotide resolution profiling of m(3)C RNA modification by HAC-seq

Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called ‘epitranscriptome’, contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m(3)C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m(3)C throughout the transcriptome are lacking. Here, we developed a m(3)C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m(3)C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m(3)C-modified RNA species, with 17 m(3)C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m(3)C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m(3)C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m(3)C methylome in different cells and tissues.