Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2-4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2-8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.
Bibliographical noteFunding Information:
We thank Anna Haagsma, Margreet Docter, Pawel Tulinski and Inge Geuzebroek for their technical support. We thank Anna Haagsma, Mohamed Fareh, Inha Heo, Margreet Docter, and Sung Hyun Kim for their critical reading this manuscript and V. Narry Kim, Jessica Sheu-Gruttadauria, Robert A. Luenberger, and Mahipal Ganji for stimulating conversations. I.J.M. and N.T.S. were supported by NIH grant R01 GM104475. C.J. was funded by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement n° (309509).
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