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

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)125-132
Number of pages8
JournalMolecular Cell
Volume59
Issue number1
DOIs
Publication statusPublished - 2015 Jan 1
Externally publishedYes

Fingerprint

RNA-Induced Silencing Complex
RNA Interference
Drosophila
Seeds
Enzymes
RNA Cleavage
Complementary RNA
Base Pairing
Small Interfering RNA
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex. / Yao, Chunyan; Sasaki, Hiroshi M.; Ueda, Takuya; Tomari, Yukihide; Tadakuma, Hisashi.

In: Molecular Cell, Vol. 59, No. 1, 01.01.2015, p. 125-132.

Research output: Contribution to journalArticle

Yao, Chunyan ; Sasaki, Hiroshi M. ; Ueda, Takuya ; Tomari, Yukihide ; Tadakuma, Hisashi. / Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex. In: Molecular Cell. 2015 ; Vol. 59, No. 1. pp. 125-132.
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