RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Alternative pre-mRNA splicing is crucial for tissue-specific gene expression and protein diversity.
• Cooperative regulation by multiple splicing factors is common, yet the structural mechanisms of RNA recognition remain unclear.
• In Caenorhabditis elegans, fibroblast growth factor receptor (FGFR) specificity relies on alternative splicing of the egl-15 gene.

Purpose of the Study:

• To elucidate the structural basis of cooperative RNA recognition by splicing factors.
• To understand how RBFOX proteins (ASD-1 and SUP-12) interact with their target RNA from the egl-15 gene.
• To validate the functional significance of this interaction in vivo.

Main Methods:

• Solution structure determination of a ternary complex involving RNA-recognition motif (RRM) domains of ASD-1, SUP-12, and target RNA.
• Multichromatic fluorescence splicing reporters to assess splicing regulation in vivo.
• Bioinformatic search for new RNA targets of the protein complex.

Main Results:

• The study determined the solution structure of a ternary complex formed by the RRM domains of ASD-1, SUP-12, and egl-15 RNA.
• The two RRM domains cooperatively bind the RNA by sandwiching a guanine base, forming a stable complex.
• In vivo experiments confirmed the necessity of the guanine base and the specific arrangement of cis elements for effective splicing regulation.

Conclusions:

• The structural mechanism of cooperative RNA recognition by RBFOX proteins involves direct interaction with a specific base (guanine).
• This cooperative binding is essential for the in vivo regulation of alternative splicing of the FGFR gene egl-15.
• The findings provide a structural foundation for understanding splicing factor cooperation and identify new functional targets.