Clusters of deep intronic RbFox motifs regulate mammalian alternative splicing

The RbFox RNA binding proteins regulate alternative splicing of genes governing mammalian development and organ function. They bind to the RNA sequence GCAUG with high affinity but also non-canonical secondary motifs in a concentration dependent manner. However, the hierarchical requirement of RbFox motifs, which are widespread in the genome, is still unclear. Here we show that deep intronic, tightly clustered RbFox1 motifs cooperate and are necessary in regulating splicing of alternative exons. By employing recombineering mutagenesis in bacterial artificial chromosomes we found that tightly clustered (U)GCAUG motifs in the middle of 50 Kb introns are necessary for RbFox regulation of gene isoforms expression. Bioinformatic analysis revealed that (U)GCAUG-clusters are widely present in both mouse and human genes and are embedded in sequences binding the large assembly of splicing regulators. Integrative data analysis from eCLIP and RNAseq experiments showed a global increase in RNA isoform modulation of genes with Rbfox1 eCLIP-peaks associated with these clusters. These data suggest that clustered distal intronic Rbfox-binding motifs are important determinants of RbFox1 function in the mammalian genome and provide a target for identification of pathogenic mutations.