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| Status |
Public on Apr 21, 2013 |
| Title |
Identification of direct targets and modified bases of RNA cytosine methyltransferases |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
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.
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| 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.
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| Contributor(s) |
Khoddami V, Cairns BR |
| Citation(s) |
23604283 |
| Submission date |
Jun 27, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Vahid Khoddami |
| Organization name |
Huntsman Cancer Institute-University of Utah
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| Department |
Oncological Sciences
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| Lab |
Bradley R Cairns
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| Street address |
4350, 2000 Circle of Hope, HCI
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| City |
SLC |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (7)
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| Relations |
| BioProject |
PRJNA169525 |
| SRA |
SRP013942 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE38957_01-DNMT2_Aza-IP_tRNAs_and_signature_analysis.xls.gz |
227.1 Kb |
(ftp)(http) |
XLS |
| GSE38957_02-DNMT2_Aza-IP_All_Genes.xls.gz |
4.7 Mb |
(ftp)(http) |
XLS |
| GSE38957_03-NSUN2_Aza-IP_All_Genes.xls.gz |
2.7 Mb |
(ftp)(http) |
XLS |
| GSE38957_04-NSUN2_Aza-IP_VarScan_signature_analysis.xls.gz |
1.7 Mb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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