Identification of Direct Targets and Modified Bases of RNA Cytosine Methyltransferases

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2013 Apr 23

PMID 23604283

Citations 244

Authors

Vahid Khoddami

Bradley R Cairns

Affiliations

Soon will be listed here.

Abstract

The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and noncoding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C→G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of RNA cytosine methyltransferases, Aza-IP may be generally applicable for target identification.

Citing Articles

Detection, molecular function and mechanisms of m5C in cancer.

Zhang L, Li Y, Li L, Yao F, Cai M, Ye D Clin Transl Med. 2025; 15(3):e70239.

PMID: 40008496 PMC: 11862898. DOI: 10.1002/ctm2.70239.

Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress.

Dougherty S, Barros G, Foster M, Teo G, Choi H, Silva G bioRxiv. 2025; .

PMID: 39975283 PMC: 11838502. DOI: 10.1101/2024.05.02.592277.

Advanced reactivity-based sequencing methods for mRNA epitranscriptome profiling.

Cai Z, Song P, Yu K, Jia G RSC Chem Biol. 2025; 6(2):150-169.

PMID: 39759443 PMC: 11694185. DOI: 10.1039/d4cb00215f.

Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins.

Ivancova I, Quirante T, Ondrus M, Pohl R, Vlkova M, Zilecka E Commun Chem. 2025; 8(1):1.

PMID: 39748090 PMC: 11696893. DOI: 10.1038/s42004-024-01399-6.

Exploration of the mechanism of 5-Methylcytosine promoting the progression of hepatocellular carcinoma.

Zhang Q, Cao Z, He Y, Liu Z, Guo W Transl Oncol. 2024; 52():102257.

PMID: 39733743 PMC: 11743813. DOI: 10.1016/j.tranon.2024.102257.

References

Motorin Y, Grosjean H . Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme. RNA. 1999; 5(8):1105-18. PMC: 1369833. DOI: 10.1017/s1355838299982201. View

Guelorget A, Golinelli-Pimpaneau B . Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes. Structure. 2011; 19(3):282-91. DOI: 10.1016/j.str.2011.01.005. View

Auxilien S, Guerineau V, Szweykowska-Kulinska Z, Golinelli-Pimpaneau B . The human tRNA m (5) C methyltransferase Misu is multisite-specific. RNA Biol. 2012; 9(11):1331-8. PMC: 3597573. DOI: 10.4161/rna.22180. View

Hussain S, Benavente S, Nascimento E, Dragoni I, Kurowski A, Gillich A . The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability. J Cell Biol. 2009; 186(1):27-40. PMC: 2712989. DOI: 10.1083/jcb.200810180. View

Hu S, Wu J, Chen L, Shan G . Signals from noncoding RNAs: unconventional roles for conventional pol III transcripts. Int J Biochem Cell Biol. 2012; 44(11):1847-51. DOI: 10.1016/j.biocel.2012.07.013. View

Chan P, Lowe T . GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 2008; 37(Database issue):D93-7. PMC: 2686519. DOI: 10.1093/nar/gkn787. View

Santi D, Garrett C, Barr P . On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell. 1983; 33(1):9-10. DOI: 10.1016/0092-8674(83)90327-6. View

Motorin Y, Lyko F, Helm M . 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res. 2009; 38(5):1415-30. PMC: 2836557. DOI: 10.1093/nar/gkp1117. View

Schaefer M, Pollex T, Hanna K, Tuorto F, Meusburger M, Helm M . RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. Genes Dev. 2010; 24(15):1590-5. PMC: 2912555. DOI: 10.1101/gad.586710. View

10.

Koboldt D, Chen K, Wylie T, Larson D, McLellan M, Mardis E . VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics. 2009; 25(17):2283-5. PMC: 2734323. DOI: 10.1093/bioinformatics/btp373. View

11.

Becker M, Muller S, Nellen W, Jurkowski T, Jeltsch A, Ehrenhofer-Murray A . Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling. Nucleic Acids Res. 2012; 40(22):11648-58. PMC: 3526270. DOI: 10.1093/nar/gks956. View

12.

Tuorto F, Liebers R, Musch T, Schaefer M, Hofmann S, Kellner S . RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat Struct Mol Biol. 2012; 19(9):900-5. DOI: 10.1038/nsmb.2357. View

13.

Frye M, Watt F . The RNA methyltransferase Misu (NSun2) mediates Myc-induced proliferation and is upregulated in tumors. Curr Biol. 2006; 16(10):971-81. DOI: 10.1016/j.cub.2006.04.027. View

14.

Squires J, Patel H, Nousch M, Sibbritt T, Humphreys D, Parker B . Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 2012; 40(11):5023-33. PMC: 3367185. DOI: 10.1093/nar/gks144. View

15.

Gerard M, Myslinski E, Chylak N, Baudrey S, Krol A, Carbon P . The scaRNA2 is produced by an independent transcription unit and its processing is directed by the encoding region. Nucleic Acids Res. 2009; 38(2):370-81. PMC: 2811027. DOI: 10.1093/nar/gkp988. View

16.

Blanco S, Kurowski A, Nichols J, Watt F, Benitah S, Frye M . The RNA-methyltransferase Misu (NSun2) poises epidermal stem cells to differentiate. PLoS Genet. 2011; 7(12):e1002403. PMC: 3228827. DOI: 10.1371/journal.pgen.1002403. View

17.

Khan M, Rafiq M, Noor A, Hussain S, Flores J, Rupp V . Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability. Am J Hum Genet. 2012; 90(5):856-63. PMC: 3376419. DOI: 10.1016/j.ajhg.2012.03.023. View

18.

Sakita-Suto S, Kanda A, Suzuki F, Sato S, Takata T, Tatsuka M . Aurora-B regulates RNA methyltransferase NSUN2. Mol Biol Cell. 2007; 18(3):1107-17. PMC: 1805108. DOI: 10.1091/mbc.e06-11-1021. View

19.

Durdevic Z, Hanna K, Gold B, Pollex T, Cherry S, Lyko F . Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2. EMBO Rep. 2013; 14(3):269-75. PMC: 3589093. DOI: 10.1038/embor.2013.3. View

20.

Yang X, Lay F, Han H, Jones P . Targeting DNA methylation for epigenetic therapy. Trends Pharmacol Sci. 2010; 31(11):536-46. PMC: 2967479. DOI: 10.1016/j.tips.2010.08.001. View