In the version of this article initially published, the data in Fig. 6h and Extended Data Fig. 6j were incorrect due to errors in the simulation code. The correct figures are shown below. The Fig. 6h caption has been updated to “Arrows indicate unmasking time of the best fit.”, as the panel now contains more than one arrow. The Extended Data Fig. 6j caption incorrectly stated “Simulated cleavage curves for 10 and 0.1 nM siRNA concentration using fast or slow unmasking rates (average unmasking time of 1 s and 800 s, respectively).” The correct average unmasking time for 0.1 nM siRNA is 1,200 s. The errors have been corrected in the HTML and PDF versions of the article. (Figure presented.).

Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by guiding Argonaute2 (AGO2) to its target site. Target site accessibility is critical for AGO2-target interactions, but how target site accessibility is controlled in vivo is poorly understood. Here, we use live-cell single-molecule imaging in human cells to determine rate constants of the AGO2 cleavage cycle in vivo. We find that the rate-limiting step in mRNA cleavage frequently involves unmasking of target sites by translating ribosomes. Target site masking is caused by heterogeneous intramolecular RNA-RNA interactions, which can conceal target sites for many minutes in the absence of translation. Our results uncover how dynamic changes in mRNA structure shape AGO2-target recognition, provide estimates of mRNA folding and unfolding rates in vivo, and provide experimental evidence for the role of mRNA structural dynamics in control of mRNA-protein interactions.