TY  - BOOK
AU  - Khoddami,V.
AU  - Cairns,B.R.
TI  - Transcriptome-wide target profiling of RNA cytosine methyltransferases using the mechanism-based enrichment procedure Aza-IP
KW  - AZACITIDINE
KW  - CYTOSINE
KW  - GENE EXPRESSION PROFILING
KW  - HIGH-THROUGHPUT NUCLEOTIDE SEQUENCING
KW  - HUMANS
KW  - IMMUNOPRECIPITATION
KW  - TRNA METHYLTRANSFERASES
N2  - 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
UR  - https://drive.google.com/file/d/13F9rvYwG6vF8-8UZqI_Y0ma5isnDV4Ok/view?usp=drivesdk
ER  -