Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo

Zhiyong Yang, Giedrius Vilkaitis, Bin Yu, Saulius Klimašauskas, Xuemei Chen

Research output: Chapter in Book/Report/Conference proceedingChapter

25 Scopus citations

Abstract

The biogenesis of microRNAs (miRNAs) in plants is similar to that in animals, however, the processing of plant miRNAs consists of an additional step, the methylation of the miRNAs on the 3′ terminal nucleotides. The enzyme that methylates Arabidopsis miRNAs is encoded by a gene named HEN1, which has been shown genetically to be required for miRNA biogenesis in vivo. Small interfering RNAs (siRNAs) are also methylated in vivo in a HEN1-dependent manner. Our biochemical studies demonstrated that HEN1 is a methyltransferase acting on both miRNAs and siRNAs in vitro. HEN1 recognizes 21 to 24 nt small RNA duplexes, which are the products of Dicer-like enzymes, and transfers a methyl group from S-adenosylmethionine (SAM) to the 2′ OH of the last nucleotides of the small RNA duplexes. Here we describe methods to characterize the biochemical activities of the HEN1 protein both in vitro and in vivo, and methods to analyze the methylation status of small RNAs in vivo.

Original languageEnglish (US)
Title of host publicationMicroRNA Methods
EditorsJohn Rossi, Gregory Hannon
Pages139-154
Number of pages16
DOIs
StatePublished - Dec 1 2007

Publication series

NameMethods in Enzymology
Volume427
ISSN (Print)0076-6879

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Yang, Z., Vilkaitis, G., Yu, B., Klimašauskas, S., & Chen, X. (2007). Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo. In J. Rossi, & G. Hannon (Eds.), MicroRNA Methods (pp. 139-154). (Methods in Enzymology; Vol. 427). https://doi.org/10.1016/S0076-6879(07)27008-9