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

Area of Science:

• Molecular Biology
• Genetics
• RNA Biology

Background:

• RbFox RNA binding proteins are critical regulators of alternative splicing in mammalian development and organ function.
• RbFox proteins bind specific RNA sequences ((U)GCAUG) with high affinity and secondary motifs concentration-dependently.
• The precise role and hierarchy of widespread RbFox motifs in gene regulation remained unclear.

Purpose of the Study:

• To investigate the function of clustered RbFox motifs in regulating alternative splicing.
• To determine the hierarchical requirement of RbFox motifs in deep intronic regions.
• To explore the interaction of RbFox motifs with other splicing regulators like LASR.

Main Methods:

• Bioinformatic analysis of (U)GCAUG clusters in mouse and human genes.
• Integrative analysis of eCLIP and RNAseq data to identify Rbfox1 targets.
• Recombineering mutagenesis in a bacterial artificial chromosome to study NTrk2 gene splicing regulation.

Main Results:

• Deep intronic, tightly clustered RbFox1 motifs were identified as key regulators of alternative exon splicing.
• (U)GCAUG clusters are prevalent in mammalian genes and embedded within sequences binding the large assembly of splicing regulators (LASR).
• Rbfox1 binding to these clusters globally increased RNA isoform modulation, and clustered motifs were essential for NTrk2 alternative splicing regulation.

Conclusions:

• Clustered, intronic RbFox-binding motifs, particularly within LASR-binding sequences, are critical determinants of RbFox1 function.
• These clustered motifs may facilitate RbFox protein recruitment to form a splicing-regulatory complex.
• The findings offer insights into the mechanisms of alternative splicing and potential targets for pathogenic mutation identification.