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.
Bibliographical noteFunding Information:
We thank M. Depken for helpful discussions with the computational modeling. We thank L. Steller, I. Bally, and R. Banerjee for help with experiments. We would also like to thank the Tanenbaum lab members for helpful discussions and T. Hoek and D. Khuperkar for critical reading of the manuscript. This work was financially supported by the European Research Council (ERC) through an ERC starting grant (ERCSTG 677936-RNAREG) to M.E.T., a VENI grant from the Netherlands Organization for Scientific Research (NWO) (NWO 016.VENI.171.050) to S.R., an ERC consolidator grant (819299) and a VIDI grant from NWO (864.14.002) to C.J., and the National Institute of General Medical Sciences (R35 GM127090) to I.J.M.; M.E.T., S.R., S.S., D.d.S. and I.L. are supported by the Oncode Institute that is partly funded by the Dutch Cancer Society (KWF).
© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.