The RNAi-mediated target cleavage reaction was analyzed by single-molecule imaging
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The seed base paring is disproportionally stabilized throughout the reaction
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Target recognition proceeds via 5′ → 3′ base-pairing propagation with proofreading
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Cleaved fragments are released independently based on the sequence and stability
Summary
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.
