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Analysis of Methylation Status of Plant MicroRNAs

  • Susu Chen
  • Guodong RenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

Small noncoding RNAs of 20–30 nucleotides in length are key mediators of gene silencing. 2′-O-Methylation on the 3′ terminal nucleotide of several types of small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants, PIWI-interacting RNAs (piRNAs) in animals, and some siRNAs in Drosophila and Caenorhabditis elegans, provides a key protective mechanism against 3′ tailing- and trimming-mediated destabilization. The methylation reaction is catalyzed by the small RNA methyltransferase HUA ENHANCER 1 (HEN1). In this chapter, we describe a detailed protocol for analyzing 3′ end methylation status of plant miRNAs, which can be applicable to other types of small RNAs as well.

Key words

MicroRNA HEN1 Methylation β-elimination Denaturing PAGE Northern blot 

Notes

Acknowledgment

Work in the Ren laboratory is supported by grants from the National Key R&D Program of China (2016YFA0503200) and the National Natural Science Foundation of China (91740101, 31622009 and 31471221).

No conflict of interest was declared.

References

  1. 1.
    D’Ario M, Griffiths-Jone S, Kim M (2017) Small RNAs: big impact on plant development. Trends Plant Sci 22:1056–1068CrossRefGoogle Scholar
  2. 2.
    Ren G, Chen X, Yu B (2014) Small RNAs meet their targets: when methylation defends miRNAs from uridylation. RNA Biol 11:1099–1104CrossRefGoogle Scholar
  3. 3.
    Yu B, Yang Z, Li J et al (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307:932–935CrossRefGoogle Scholar
  4. 4.
    Huang Y, Ji L, Huang Q et al (2009) Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461:823–827CrossRefGoogle Scholar
  5. 5.
    Yang Z, Ebright YW, Yu B et al (2006) HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res 34:667–675CrossRefGoogle Scholar
  6. 6.
    Saito K, Sakaguchi Y, Suzuki T et al (2007) Pimet, the Drosophila homolog of HEN1, mediates 2′-O-methylation of Piwi- interacting RNAs at their 3′ ends. Genes Dev 21:1603–1608CrossRefGoogle Scholar
  7. 7.
    Horwich MD, Li C, Matranga C et al (2007) The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr Biol 17:1265–1272CrossRefGoogle Scholar
  8. 8.
    Kirino Y, Mourelatos Z (2007) The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs. RNA 13:1397–1401CrossRefGoogle Scholar
  9. 9.
    Li J, Yang Z, Yu B et al (2005) Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr Biol 15:1501–1507CrossRefGoogle Scholar
  10. 10.
    Zhao Y, Yu Y, Zhai J et al (2012) The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation. Curr Biol 22:689–694CrossRefGoogle Scholar
  11. 11.
    Ren G, Chen X, Yu B (2012) Uridylation of miRNAs by HEN1 SUPPRESSOR1 in Arabidopsis. Curr Biol 22:695–700CrossRefGoogle Scholar
  12. 12.
    Tu B, Liu L, Xu C et al (2015) Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis. PLoS Genet 11:e1005119CrossRefGoogle Scholar
  13. 13.
    Wang X, Zhang S, Dou Y et al (2015) Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3′ tailing of small RNAs in Arabidopsis. PLoS Genet 11:e1005091CrossRefGoogle Scholar
  14. 14.
    Zhai J, Zhao Y, Simon SA et al (2013) Plant microRNAs display differential 3′ truncation and tailing modifications that are ARGONAUTE1 dependent and conserved across species. Plant Cell 25:2417–2428CrossRefGoogle Scholar
  15. 15.
    Tsai HL, Li YH, Hsieh WP et al (2014) HUA ENHANCER1 is involved in posttranscriptional regulation of positive and negative regulators in Arabidopsis photomorphogenesis. Plant Cell 26:2858–2872CrossRefGoogle Scholar
  16. 16.
    Alefelder S, Patel BK, Eckstein F (1998) Incorporation of terminal phosphorothioates into oligonucleotides. Nucleic Acids Res 26:4983–4988CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Genetic Engineering and Institute of Plant Biology, Department of Biochemistry, School of Life SciencesFudan UniversityShanghaiChina

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