TY - JOUR
T1 - Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex
AU - Yao, Chunyan
AU - Sasaki, Hiroshi M.
AU - Ueda, Takuya
AU - Tomari, Yukihide
AU - Tadakuma, Hisashi
N1 - Funding Information:
We thank Kaori Kiyokawa for the maintenance and propagation of S2 cells and the members of our laboratory for discussions and critical comments on the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (“Functional machinery for non-coding RNAs” and “non-coding RNA neo-taxonomy”) (to H.T. and Y.T.), a Grant-in-Aid for Young Scientists (A) (to H.T.), a Grant-in-Aid for Challenging Exploratory Research (to H.M.S.), a Japan-China Sasakawa Medical Fellowship from The Nippon Foundation and National Natural Science Foundation of China (81371885, to C.Y.), and a Research Award from the Takeda Science Foundation (to Y.T.).
Publisher Copyright:
© 2015 Elsevier Inc.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
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U2 - 10.1016/j.molcel.2015.05.015
DO - 10.1016/j.molcel.2015.05.015
M3 - Article
C2 - 26140368
AN - SCOPUS:84937124444
SN - 1097-2765
VL - 59
SP - 125
EP - 132
JO - Molecular Cell
JF - Molecular Cell
IS - 1
ER -