The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Expression

Background:

• Alternative splicing is crucial for gene expression regulation.
• Proteins like ASF/SF2 and SC35 are essential for pre-mRNA splicing and alternative splice site selection.

Purpose of the Study:

• To investigate if ASF/SF2 and SC35 exhibit distinct RNA binding specificities.
• To determine if these specificities influence their functional interactions with pre-mRNA.
• To elucidate the role of these proteins in splicing enhancer function.

Main Methods:

• Selection/amplification of random RNA sequences (SELEX) using RNA-binding domains (RBDs) of ASF/SF2 and SC35.
• Binding assays with full-length proteins to confirm specificity.
• Demonstration of splicing enhancer activity using multi-copy binding site elements.

Main Results:

• Both ASF/SF2 and SC35 selected purine-rich sequences, but with distinct motifs.
• Full-length proteins confirmed different specificities; arginine-serine regions were not major determinants.
• Cooperation between ASF/SF2's two RBDs contributes to its binding specificity.
• An element with three high-affinity ASF/SF2 binding sites acted as a potent splicing enhancer, while a similar SC35 element was inactive.
• ASF/SF2 enhancer activity was dependent on recombinant ASF/SF2 and additional protein factors in splicing-deficient extracts.

Conclusions:

• ASF/SF2 and SC35 possess distinct RNA binding specificities.
• ASF/SF2 plays a central role in the function of purine-rich splicing enhancers.
• The differential binding specificities and enhancer activities suggest distinct functional roles for ASF/SF2 and SC35 in alternative splicing regulation.