A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing [CLEAR-CLIP]

miRNAs are regulatory transcripts established as repressors of mRNA stability and translation. Here we demonstrate that an oncomiR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of direct binding between miR-10b and U6, in situ visualizations of miR-10b and U6 co-localization in glioma cells and tumor tissues, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. The effects on U6 result in splicing alterations, illustrated by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a cancer-associated miRNA. Overall design: To investigate endogenous miR-10b targets, we employed the modified CLEAR- CLIP protocol in glioma cells (Moore et al., 2015). The technique relies on miRNA crosslinking with proximal transcripts, ligation, and sequencing of chimeras. Therefore, it provides an unbiased mapping of the miRNA-target interactions, independent of bioinformatic predictions.