Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Alternative splicing (AS) generates transcriptome complexity, regulated by RNA binding proteins (RBPs).
• Muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) proteins are key regulators of developmental AS transitions.
• Understanding RBP concentration effects on AS is crucial for developmental biology.

Purpose of the Study:

• To investigate how RNA binding protein concentrations influence alternative splicing transcriptome-wide.
• To characterize the dose-dependent effects of MBNL1 on skipped exon events.

Main Methods:

• Engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line for controlled RBP expression.
• Performed focused analysis of dose-dependent MBNL1 effects on skipped exon alternative splicing.
• Generated transcriptome-wide dose-response curves to analyze splicing regulation.

Main Results:

• Modest RBFOX1 induction modulated MBNL1-dependent AS despite endogenous RBFOX levels.
• MBNL1-regulated exclusion events may require higher protein concentrations than inclusion events.
• Multiple YGCY motif arrangements can yield similar splicing outcomes, indicating complex regulation.

Conclusions:

• Alternative splicing regulation involves complex interaction networks across RBP gradients, not just simple binding site arrangements.
• The concentration of RBPs significantly impacts specific AS outcomes, including inclusion and exclusion events.
• This study provides insights into the nuanced control of gene expression through alternative splicing.