Transcriptome-wide target profiling of RNA cytosine methyltransferases using the mechanism-based enrichment procedure Aza-IP 

 -  Nature Protocols, 9(2), p.337-361, 2014 .
<br/><br/> 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. © 2014 Nature America, Inc. All rights reserved. 
<br/><br/><label>Subjects--Topical Terms: </label><br/>AZACITIDINE<br/>CYTOSINE<br/>GENE EXPRESSION PROFILING<br/>HIGH-THROUGHPUT NUCLEOTIDE SEQUENCING<br/>HUMANS<br/>IMMUNOPRECIPITATION<br/>TRNA METHYLTRANSFERASES