Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin-associated RNA and silence gene expression.

The action of microRNAs (miRNAs) in mammalian cells involves recognition of messenger RNA (mRNA) in the cytoplasm and inhibition of translation. Both miRNAs and their associated protein factors, 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 regulation. 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 both the cytoplasm and the nucleus. let-7 had little effect on HMGA2 transcription or splicing. Our data validate chimeric eCLIP as a powerful method for experimentally identifying promising miRNA:RNA interactions. Rather than a solely cytoplasmic event, binding of miRNA-associated protein factors to mRNA targets may begin in the nucleus. Gene silencing reduces RNA levels in both the cytoplasm and the nucleus. miRNA-mediated silencing of mRNAs may be influenced by both nuclear and cytoplasmic interactions.