Global analysis of AGO2-bound RNAs in teratoma-derived fibroblasts

Among the four Argonaute family members in mammals, only AGO2 protein retains endonuclease activity, and facilitates cleavage of target RNAs base-pairing with highly complementary guide RNAs. Despite the deeply conserved catalytic activity, only a small number of targets have been reported to extensively base pair with cognate miRNAs to be cleaved by AGO2. Here, we analyzed AGO2-bound RNAs by CrossLinking ImmunoPrecipitation (CLIP) of genetically modified cells that express epitope-tagged AGO2 from the native genomic locus. We found that HMGA2 mRNA is cleaved by AGO2 loaded with let-7 and miR-21. In contrast to the generally accepted notion, the base-pairing from the seed region to the cleavage site, rather than perfect or near perfect complementarity, was required for cleavage of the target mRNA in cells. Non-templated addition of nucleotides at the 3’ end of the cleaved RNA was observed, further supporting the AGO2-mediated cleavage. Based on the observation that the limited complementarity is the minimum requirement for cleavage, we found that AGO2-mediated cleavage of targets is more common than previously thought. Our result may explain the vital role of endonuclease activity in controlling miRNA-mediated gene regulation.