TY - CHAP
T1 - Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo
AU - Yang, Zhiyong
AU - Vilkaitis, Giedrius
AU - Yu, Bin
AU - Klimašauskas, Saulius
AU - Chen, Xuemei
N1 - Funding Information:
We thank Dr. Yon W. Ebright for her help with the HPLC analysis, Drs. Manu Agarwal and Vanitharani Ramachandran for comments on the manuscript, and Alexandra Plotnikova for technical assistance. This work was funded by a National Science Foundation grant (MCB 0343480) to X. C. The work of G. V. was supported by an EU Centers of Excellence program grant (QLK3‐CT2002‐30575).
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
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U2 - 10.1016/S0076-6879(07)27008-9
DO - 10.1016/S0076-6879(07)27008-9
M3 - Chapter
C2 - 17720483
AN - SCOPUS:42949086521
SN - 9780123739179
T3 - Methods in Enzymology
SP - 139
EP - 154
BT - MicroRNA Methods
A2 - Rossi, John
A2 - Hannon, Gregory
ER -