GSE38957
Identification of direct targets and modified bases of RNA cytosine methyltransferases
We report development of a mechanism-based technique, Aza-IP, that utilizes the stable covalent linkage formed in vivo between an RNA methyltransferase (m5C-RMT) and 5-azacytidine (5-aza-C) incorporated within the target RNA to enable specific target enrichment, coupled with high-throughput sequencing to provide target identification. We apply Aza-IP to two enzyme types, DNMT2 and NSUN2, the latter with important roles in fertility, intellectual ability, stem cells and cancer. For both, Aza-IP provided >200-fold enrichment of their known human tRNA targets. For NSUN2, we reveal many novel tRNA and non-coding RNA targets, all linked to NSUN2 nucleolar localization, greatly increasing the potential repertoire underlying loss-of-function pathologies.  Strikingly, we observe a C>G transversion ‘signature’ specifically at the target cytosine, which enables the identification of the exact target cytosine within the RNA in the same experiment. Overall design: The general design of Aza-IP involves nine steps: 1) Expression of an epitope-tagged m5C-RMT derivative (or use of an antibody capable of immuno-precipitating the RNA-bound enzyme), 2) cell growth in the presence of 5-aza-C, which incorporates at low/moderate levels into nascent RNA, 3) cell lysis, 4) immuno-precipitation of the subject m5C-RMT, a portion of which is covalently attached to target RNAs bearing 5-aza-C, 5) stringent washing to remove RNA contaminants, 6) RNA fragmentation, release and purification, 7) ligation of adaptor oligos to the RNA, and the creation of a cDNA library in a manner that enables strand-specific assignments, 8) cDNA sequencing, and 9), mapping and examination of sequence reads to define RNA targets and site of cross-linking/catalysis.