Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• RNA Splicing Mechanisms

Background:

• Alternative splicing generates protein diversity from a single gene.
• Caspase-2 pre-mRNA splicing produces anti-apoptotic (Casp-2S) and pro-apoptotic (Casp-2L) isoforms via exon 9 inclusion/skipping.
• The precise molecular mechanisms governing caspase-2 exon 9 splicing remain incompletely understood.

Purpose of the Study:

• To elucidate the role of SRSF3 (SRp20) in regulating caspase-2 pre-mRNA alternative splicing.
• To identify the specific binding sites and molecular interactions through which SRSF3 influences exon 9 splicing.
• To reveal the functional consequences of SRSF3 binding on caspase-2 pre-mRNA splicing.

Main Methods:

• RNA interference (siRNA) for SRSF3 knockdown.
• Expression vector transfection for SRSF3 overexpression.
• Site-directed mutagenesis (substitution and deletion) of potential SRSF3 binding sites.
• RNA pulldown assays and immunoblotting to confirm protein-RNA interactions.

Main Results:

• SRSF3 knockdown increased endogenous exon 9 inclusion, while SRSF3 overexpression promoted exon 9 skipping.
• Mutagenesis of a predicted SRSF3 binding site on exon 8 abolished SRSF3's regulatory effect on exon 9 splicing.
• SRSF3 directly interacts with the specific RNA sequence on exon 8, but not with mutated sequences.
• Deletions in flanking exons or introns did not affect SRSF3's regulatory function.

Conclusions:

• SRSF3 acts as a positive regulator of caspase-2 exon 9 skipping.
• The interaction of SRSF3 with a specific site on exon 8 is crucial for its function in promoting exon 9 skipping.
• This study uncovers a novel mechanism by which SRSF3 controls caspase-2 alternative splicing, impacting the balance of apoptotic and anti-apoptotic protein isoforms.