title: Transcriptome-wide target profiling of RNA cytosine methyltransferases using the mechanism-based enrichment procedure Aza-IP.
abstract: Cytosine methylation within RNA is common, but its full scope and functions are poorly understood, as the RNA targets of most mammalian cytosine RNA methyltransferases (m(5)C-RMTs) remain uncharacterized. To enable their characterization, we developed a mechanism-based method for transcriptome-wide m(5)C-RMT target profiling. All characterized mammalian m(5)C-RMTs form a reversible covalent intermediate with their cytosine substrate-a covalent linkage that is trapped when conducted on the cytosine analog 5-azacytidine (5-aza-C). We used this property to develop Aza-immunoprecipitation (Aza-IP), a methodology to form stable m(5)C-RMT-RNA linkages in cell culture, followed by IP and high-throughput sequencing, to identify direct RNA substrates of m(5)C-RMTs. Remarkably, a cytosine-to-guanine (C→G) transversion occurs specifically at target cytosines, allowing the simultaneous identification of the precise target cytosine within each RNA. Thus, Aza-IP reports only direct RNA substrates and the C→G transversion provides an important criterion for target cytosine identification, which is not available in alternative approaches. Here we present a step-by-step protocol for Aza-IP and downstream analysis, designed to reveal identification of substrate RNAs and precise cytosine targets of m(5)C-RMTs. The entire protocol takes 40-50 d to complete.
authors.affiliation: Howard Hughes Medical Institute (HHMI), 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.affiliation: Howard Hughes Medical Institute (HHMI), Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Cairns
mesh_terms: D001374:Azacitidine / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D012359:tRNA Methyltransferases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003596:Cytosine; D012359:tRNA Methyltransferases; D001374:Azacitidine