Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin and silence gene expression

RNA interference (RNAi) in mammalian cells involves recognition of mRNA in the cytoplasm and inhibition of translation. Both protein RNAi factors and miRNAs, however, are present in mammalian cell nuclei. It is unclear how this nuclear localization affects endogenous gene expression. Here, we use chimeric eCLIP to identify complexes of Argonaute 2 (AGO2) and miRNAs. We identify the most abundant miRNAs associated with chromatin and their chromatin-associated RNA targets. Chimeric eCLIP revealed that High mobility group AT-Hook 2 (HMGA2) was the most compelling target for miRNA-mediated gene binding. There are four confirmed let-7 miRNA sites within the 3'-UTR in the cytoplasm or nucleus and three within chromatin-associated RNA. The expression of mature HMGA2 mRNA was repressed by let-7 in the cytoplasm and nucleus. let-7 had little effect splicing or transcription. Our data validate chimeric eCLIP as a powerful method for experimentally identifying promising miRNA:RNA interactions. Rather than a solely cytoplasmic event, binding of RNAi factors to mRNA targets may begin in the nucleus through a mechanism that can reduce RNA levels in both the cytoplasm and the nucleus. miRNA-mediated silencing of mRNAs may be influenced by both nuclear and cytoplasmic interactions. Overall design: Argonaute 2 (AGO2) miR-eCLIP in HCT116 whole cell, cytoplasm and chromatin, and HCT116 NLS-AGO2 whole cell samples.