Identification of direct targets and modified bases of RNA cytosine methyltransferases

Abstract

The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a mechanism-based technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and non-coding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C>G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of cytosine RNA methyltransferases, Aza-IP may be generally applicable for target identification.

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

“…A few studies have also utilized RNA bisulfite sequencing to map RNA m5C marks at the transcriptome level and found evidence for the presence of m5C in mammalian mRNAs and noncoding RNAs (Squires et al 2012;Hussain et al 2013b;Khoddami and Cairns 2013;Amort et al 2017). While it has been suggested that the human coding and noncoding transcriptome contains up to 10,000 methylation sites (Squires et al 2012;Amort et al 2017), the function of these marks has remained elusive.…”

The RNA modification landscape in human disease

“…Recent studies have provided detailed maps of the location and abundance of a handful of RNA modifications (Dominissini et al 2012;Meyer et al 2012;Khoddami and Cairns 2013;Carlile et al 2014;Schwartz et al 2014a;Delatte et al 2016), mostly obtained by coupling antibody immunoprecipitation or chemical treatments to next-generation sequencing. Through these approaches, N 6 -methyladenosine (m 6 A) modification has been identified as an important factor in the determination of mammalian cell fate transition and embryonic stem cell differentiation (Batista et al 2014;.…”

Publisher's Note

“…3 Mapping of m 5 C in RNA was initially attempted by adapting the bisulfite sequencing technology used for m 5 C in DNA, leading to the identification of approximately 10,000 m 5 C sites in mRNA. 26,27 Subsequently, new methods were developed that leverage m 5 C methyltransferases to enrich m 5 C-containing RNA, either by incorporating 5-azacytidine into RNA, 28 or by mutating the methyltransferase 29 to enable modified RNA capture. These methods identified far fewer m 5 C sites in mRNA, on the order of a few hundred to one thousand, respectively.…”