Sites for Argonaute Binding within Human Nuclear RNA and their Impact on Alternative Splicing

Mammalian RNA interference (RNAi) is often linked to regulation in the cytoplasm. While synthetic RNAs regulate transcription and splicing, endogenous functions for nuclear RNAi have been obscure. Using enhanced crosslinking immunoprecipitation (eCLIP), we mapped AGO2 binding sites within nuclear RNA. The strongest AGO binding sites mapped to miRNAs. The prevalence of individual miRNAs was similar in the cytoplasm and the nucleus. Most statistically-significant AGO2 binding was within intron boundaries. RNAseq comparing AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout to wild-type cells revealed that binding was associated with both up- and down-regulation of gene expression. We observed changes of splicing at genes with significant AGO binding at exon/intron junctions or within introns. Splicing changes were confirmed by RT-PCR and could be recapitulated by synthetic duplex RNAs that target the sites of AGO binding. Inhibition of miRNA action by addition of an anti-miR reversed miRNA-mediated control of splicing. We conclude: 1) That AGO binding sites occur throughout nuclear RNA but are often within introns; 2) The distribution of miRNAs is similar in the nucleus and cytoplasm; and 3) miRNAs can control gene splicing. Nuclear RNAi has the potential to be a natural regulatory mechanism but careful study will be necessary to identify critical RNA drivers of normal physiology and disease. Overall design: Total 23 samples, including 15 for standard transcriptome-type RNA-seq and 8 for eCLIP-seq