A RRM1-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion

RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3’ intronic sites only. Here we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognises a GGA-centred motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognises a C-rich sequence, which explains its known interaction with the intronic 3’ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10’s broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping. In the RNAcompete assay, purified GST-tagged RBM10 proteins (RRM1-ZnF and RRM1-ZnF-RRM2) are incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.