HAC‐seq: A m 3 C‐Specific Sequencing Technique for Nucleotide‐Resolution Profiling of m 3 C Methylome on RNA

Abstract

Objective 3-methylcytidine (m3C) modification is a poorly studied RNA modification. Methods for the specific mapping of m3C throughout the transcriptome are lacking. The objective of this study is to develop a m3C-specific sequencing technique to profile the m3C methylome at single-nucleotide resolution. Methods and Results Previous studies have shown that hydrazine can specifically induce nucleophilic addition for m3C residues in RNA under high salt conditions. We make use of this old m3C-specific chemical reaction to detect m3C on RNA. Since tRNAs are the mostly extensively studied cellular RNA species for m3C modification, we tested our method by using isolated small RNA (smRNA) (< 200 nt). We first incubated smRNAs with 10% hydrazine with 3M NaCl. Then RNAs were subjected to aniline-induced cleavage of the RNA chain. HPLC-MS/MS analysis showed that hydrazine/aniline treatment was able to decrease m3C levels on RNA. Additionally, northern blot analysis supported that hydrazine/aniline treatment was able to generate one or two cleaved 3’ fragments of the correct sizes for several known m3C-modified tRNAs. We further found that demethylation treatment by AlkB was able to diminish the hydrazine/aniline induced cleavage on tRNA. All demonstrate that hydrazine/aniline treatment can specifically cleave tRNAs at m3C modification sites. In order to explore the global m3C RNA methylome we then coupled this hydrazine/aniline-induced chemical cleavage method with next generation sequencing to establish a Hydrazine-Aniline Cleavage sequencing (HAC-seq) technique for the identification of m3C modification sites on RNAs at single-nucleotide resolution. rRNA-depleted total RNAs were randomly fragmented and end-repaired. The fragmented RNAs were treated with 10% hydrazine with 3M NaCl followed by aniline to induce the cleavage of the RNA backbone at the m3C modification sites. The 5’ fragment generated by HAC contains a damaged 3’ end without the correct 3’-OH group which prevents the adaptor ligation in the library preparation step. Only the full length and 3’ cleaved fragments can be subsequently sequenced. After the bioinformatic data analysis, m3C-modified sites are determined by calculating the Cleavage Ratio at single nucleotide resolution. HAC-seq revealed that tRNAs are the predominant m3C-modified RNA species, with 17 different m3C sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs. In addition, the cleavage ratio calculated from HAC-seq can be used to estimate m3C levels on RNA. Overall, cytoplasmic and mitochondrial tRNA-Thr species are the highest-m3C modified tRNAs. Conclusions HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution. The cleavage ratio calculated from HAC-seq can be used to estimate m3C levels on RNA.