Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• The Rbfox family of splicing factors are crucial regulators of alternative splicing in animals.
• Rbfox proteins are known to bind the GCAUG motif with high affinity.
• However, only half of in vivo Rbfox binding sites contain this canonical motif.

Purpose of the Study:

• To identify and characterize non-canonical Rbfox binding sites.
• To investigate the functional significance of these secondary motifs in RNA splicing regulation.

Main Methods:

• Incubation of recombinant RBFOX2 with a large dataset of mouse and human transcriptomic sequences.
• Validation of secondary motif binding in cellular contexts.
• Assessment of regulatory capacity using a trichromatic splicing reporter assay.

Main Results:

• Discovery of substantial RBFOX2 binding to moderate-affinity, non-GCAUG sites at physiological concentrations.
• Demonstration that these secondary motifs bind Rbfox robustly in cells.
• Evidence that multiple secondary motifs can collectively regulate splicing similarly to the canonical GCAUG motif.
• Identification of secondary motif-mediated splicing regulation during neuronal development, particularly in subtypes with high Rbfox concentrations.

Conclusions:

• Rbfox proteins bind to a broader range of RNA sequences than previously known, including 'secondary motifs'.
• These secondary motifs contribute significantly to Rbfox-mediated alternative splicing, especially under conditions of elevated Rbfox levels.
• The findings reveal a more complex regulatory network for alternative splicing, impacting neuronal development and potentially disease states.