title: 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 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 noncoding 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 RNA cytosine methyltransferases, Aza-IP may be generally applicable for target identification.
authors.affiliation: Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Vahid
authors.initials: V
authors.lastname: Khoddami
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Cairns
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003596:Cytosine / Q000737:chemistry*; D047468:Immunoprecipitation / Q000379:methods*; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry* / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics
chemical_list: D012313:RNA; D003596:Cytosine; D012359:tRNA Methyltransferases