Clusters of deep intronic RbFox motifs embedded in large assembly of splicing regulators sequences regulate alternative splicing.

The RbFox RNA binding proteins regulate alternative splicing of genes governing mammalian development and organ function. They bind to the RNA sequence (U)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 important regulators of alternative exons splicing. 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 (LASR). 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. Experimentally, by employing recombineering mutagenesis in a bacterial artificial chromosome containing the NTrk2 mouse region subjected to alternative splicing we showed that tightly clustered (U)GCAUG motifs in the middle of 50 Kb introns are necessary for RbFox1 regulation of NTrk2 gene isoforms expression. Moreover, clustered (U)GCAUG-motifs promote the recruitment of RbFox1 proteins to form a Rbfox1/LASR complex required for splicing. These data suggest that clustered, distal intronic Rbfox-binding motifs embedded in LASR binding sequences are important determinants of RbFox1 function in the mammalian genome and provide a target for identification of pathogenic mutations.