Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex

Chunyan Yao, Hiroshi M. Sasaki, Takuya Ueda, Yukihide Tomari, Hisashi Tadakuma

研究成果: Article査読

32 被引用数 (Scopus)

抄録

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition.

本文言語English
ページ(範囲)125-132
ページ数8
ジャーナルMolecular Cell
59
1
DOI
出版ステータスPublished - 2015
外部発表はい

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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