MiRNA accessibility in cellular RISC revealed by single molecule kinetic fingerprinting.

Abstract

MicroRNAs (miRNAs) in association with Argonaute (Ago) proteins in the RNA induced silencing complex (RISC) regulate the levels of translation of messenger RNAs (mRNAs). The major parameter thought to explain the selection of the target mRNA is the base pairing between the ‘5’ seed’ region of the miRNA and its counterpart mRNA. Ago2 crystalizes in a conformation where the 5’ seed region is exposed to the target mRNA. However, both 5’ seed and 3’ non-seed-based target recognition has been reported. In fact, thousands of miRNA target sites depend on a non-seed pairing interaction, highlighting a gap in our understanding of miRNA targeting rules. Here, we present a ‘single molecule kinetic fingerprinting’ approach using split miRNA complementary probes (i.e., target mimics) that trace the differential accessibility of the 5’ seed and 3’ non-seed segments of the miRNA in RISC immunoprecipitated from human HeLa cell extract. Two distinct populations are observed with either the 5’ seed or the 3’ non-seed segment accessible. Only a minor population (∼5-10%) shows simultaneous accessibility of both segments. Compared to the protein-free miRNA, the target interaction kinetics of the seed and non-seed segments of the Ago2-bound miRNA are differentially controlled by the surrounding protein. In particular, tight target binding to the 5’ seed region alters the kinetics of the 3’ non-seed interaction, resulting in kinetic heterogeneity in that interaction with the target mimic. This kinetic heterogeneity most likely arises from differential accessibility of the 3’ non-seed region of miRNA in Ago2, which adopts multiple conformations. In summary, our data indicate that both 5’ seed and 3’ non-seed-based target recognition is possible, but the accessibility and the kinetics of mRNA interactions is largely governed by the surrounding protein conformation as well as the guide-target complementarity.