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 Citations (Scopus)

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

Fingerprint

Methylation
MicroRNAs
Small Interfering RNA
RNA
Nucleotides
Ribonuclease III
S-Adenosylmethionine
Methyltransferases
Enzymes
Arabidopsis
In Vitro Techniques
Animals
Genes
Processing
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

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

Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo. / Yang, Zhiyong; Vilkaitis, Giedrius; Yu, Bin; Klimašauskas, Saulius; Chen, Xuemei.

MicroRNA Methods. ed. / John Rossi; Gregory Hannon. 2007. p. 139-154 (Methods in Enzymology; Vol. 427).

Research output: Chapter in Book/Report/Conference proceedingChapter

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. Methods in Enzymology, vol. 427, pp. 139-154. https://doi.org/10.1016/S0076-6879(07)27008-9
Yang Z, Vilkaitis G, Yu B, Klimašauskas S, Chen X. Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo. In Rossi J, Hannon G, editors, MicroRNA Methods. 2007. p. 139-154. (Methods in Enzymology). https://doi.org/10.1016/S0076-6879(07)27008-9
Yang, Zhiyong ; Vilkaitis, Giedrius ; Yu, Bin ; Klimašauskas, Saulius ; Chen, Xuemei. / Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo. MicroRNA Methods. editor / John Rossi ; Gregory Hannon. 2007. pp. 139-154 (Methods in Enzymology).
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