A Bacterial Artificial Chromosome system to uncover RNA Binding Protein functions in deep intronic regions [RNA-seq]

The processing of long introns in alternative splicing of pre-mRNAs is still not well understood. Using bacterial artificial chromosome (BAC) recombineering technology, we generated a Ntrk2-BAC minigene including endogenous 50 Kb introns flanking alternatively expressed exons generating two isoforms. We found that the RNA-binding protein RbFox1 regulates the expression of the minigene-encoded isoforms. Enhanced RNA Cross-Linking Immuno-Precipitation (eCLIP), revealed that RbFox1 binds clusters of (U)GCAUG motifs present in deep intronic regions. Deletion of these clusters completely abolished RbFox1-mediated Ntrk2-isoform expression regulation. Bioinformatic analysis showed (U)GCAUG-clusters are present in both mouse and human genes. 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 BACs are powerful tools to study RNA metabolism across large genomic fragments and clustered distal intronic Rbfox motifs are important determinants of RbFox1 function in the mammalian genome providing a target for identification of intronic pathogenic mutations.