Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A-SF3b complex

Area of Science:

• Molecular biology
• RNA splicing mechanisms
• Structural biology

Background:

• Pladienolide, herboxidiene, and spliceostatin are known splicing modulators targeting SF3B1 within the SF3b complex.
• The SF3b complex is essential for pre-mRNA splicing.

Purpose of the Study:

• To investigate the role of PHF5A, a component of the SF3b subcomplex, in response to splicing modulators.
• To identify the interaction site and mechanism of action for these splicing modulators.

Main Methods:

• Site-directed mutagenesis to create PHF5A and SF3B1 variants.
• RNA sequencing (RNA-seq) to analyze splicing patterns.
• Crystal structure determination of human PHF5A.
• Cryo-electron microscopy (cryo-EM) analysis of the spliceosome B^act complex.

Main Results:

• Mutations in PHF5A (Y36) and SF3B1 (K1071, R1074, V1078) confer resistance to splicing modulators, suggesting a common interaction site.
• The PHF5A-Y36C mutation minimally affects basal splicing but globally inhibits splicing modulator activity.
• Resistance mutations cluster around the branch point adenosine in the spliceosome, indicating a competitive binding mechanism.
• PHF5A-Y36C alters splicing modulator-induced intron retention and exon skipping, correlating with GC content differences.

Conclusions:

• PHF5A is a direct target of pladienolide, herboxidiene, and spliceostatin.
• PHF5A and SF3B1 form a central binding site for these splicing modulators.
• The findings elucidate the mechanism of action for these compounds and highlight PHF5A as a key player in splicing regulation.