E-GEOD-38957 - Identification of direct targets and modified bases of RNA cytosine methyltransferases

Status
Released on 21 April 2013, last updated on 29 April 2013
Organism
Homo sapiens
Samples (7)
Protocols (4)
Description
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. 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.
Experiment type
other 
Contacts
Citation
MINSEQE
Exp. designProtocolsVariablesProcessedSeq. reads
Files
Investigation descriptionE-GEOD-38957.idf.txt
Sample and data relationshipE-GEOD-38957.sdrf.txt
Additional data (1)E-GEOD-38957.additional.1.zip
Links