RBFOX2 alters splicing outcome in distinct binding modes with multiple protein partners

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• RBFOX2 is a key regulator of RNA splicing, influencing cell differentiation and development.
• Understanding RBFOX2's regulatory mechanisms is crucial for deciphering gene expression control.

Purpose of the Study:

• To investigate the interaction partners of RBFOX2.
• To elucidate the distinct binding modes and sequence motifs RBFOX2 utilizes to regulate splicing.
• To explain the heterogeneity observed in RBFOX2-mediated splicing outcomes.

Main Methods:

• Analysis of RNA-binding protein datasets.
• RNA immunoprecipitation assays.
• RNA sequencing (RNA-seq).
• Splice-sensitive PCR.
• Knockdown of specific RNA-binding proteins (RBPs).

Main Results:

• RBFOX2 interacts with hnRNPC, hnRNPM, and SRSF1 to control a wide range of splicing events.
• RBFOX2 employs three distinct binding modes: single, multiple, and secondary.
• The single mode involves direct binding via a canonical motif; multiple mode involves co-binding with other RBPs; secondary mode involves recruitment via protein partners without direct RNA binding.
• These modes target different transcripts and influence splicing outcomes based on position and distance to alternative splice sites.

Conclusions:

• RBFOX2 utilizes diverse protein-RNA and protein-protein interactions to achieve flexible and context-dependent regulation of RNA splicing.
• The identified binding modes provide a framework for understanding the complexity and specificity of RBFOX2's role in gene expression.
• This study reveals novel insights into the intricate mechanisms governing alternative splicing and its impact on cellular processes.