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

Nature Protocols volume 9, pages 337–361 (2014)Cite this article

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Abstract

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 (m5C-RMTs) remain uncharacterized. To enable their characterization, we developed a mechanism-based method for transcriptome-wide m5C-RMT target profiling. All characterized mammalian m5C-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 m5C-RMT-RNA linkages in cell culture, followed by IP and high-throughput sequencing, to identify direct RNA substrates of m5C-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 m5C-RMTs. The entire protocol takes 40–50 d to complete.

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Figure 1: Schematic representation of 5-aza-C-mediated RNA-m5C-RMT adduct formation.
Figure 2: Schematic representation of the major steps in a typical Aza-IP experiment.
Figure 3: Aza-IP bioinformatic analysis.
Figure 4: Validation of candidate target sites by RNAi and conventional RNA bisulfite sequencing.

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Acknowledgements

We thank A. Schlichter, T. Parnell, A. Yerra, B. Dalley, N. Moss, K. Basham, D. Nix, T. Mosbruger and B. Milash for their helpful comments and suggestions for improving the manuscript. This work was supported by HHMI and the Samuel Waxman Cancer Research Foundation, as well as by the National Cancer Institute CA24014 (for core facilities). We thank V. Planelles (University of Utah) for pPR-lentiviral expression plasmid.

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Authors and Affiliations

  1. Department of Oncological Sciences, Howard Hughes Medical Institute (HHMI), Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Vahid Khoddami & Bradley R Cairns

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  1. Vahid Khoddami
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Contributions

B.R.C. and V.K. together conceived Aza-IP and the general experimental design. V.K. was involved in detailed design, implementation of all experiments and bioinformatic analysis for Aza-IP and RNA bisulfite sequencing. V.K. and B.R.C. wrote the main text. V.K. wrote the remaining text. V.K. designed and made all figures, with comments from B.R.C.

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Correspondence to Bradley R Cairns.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Schematic of the 2-step PCR amplification of NSUN2 mRNA.

First, cDNA is made using the oligo-dT primer (Step 5). Next, the F1 and R primers are used to produce amplicon 1 (Steps 6-8), which is used as template for the F2 and R primers to produce amplicon 2 (Steps 9-11). Amplicon 2 contains all the elements of the designed construct (Supplementary Data). Sequences of the F1, F2 and R primers are provided in the MATERIALS section.

Supplementary information

Supplementary Figure 1

Schematic of the 2-step PCR amplification of NSUN2 mRNA. (PDF 137 kb)

Supplementary Data

Supplementary Table 1: CDS and protein sequences of the NSUN2 variant 1. Supplementary Table 2: Designed NSUN2 amplicon for restriction enzyme digestion and cloning into lentiviral vector. (PDF 178 kb)

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Khoddami, V., Cairns, B. Transcriptome-wide target profiling of RNA cytosine methyltransferases using the mechanism-based enrichment procedure Aza-IP. Nat Protoc 9, 337–361 (2014). https://doi.org/10.1038/nprot.2014.014

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