Promoter architecture modulates CFTR exon 9 skipping

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Alternative splicing is a key mechanism for generating protein diversity.
• Promoter architecture can influence gene expression and splicing outcomes.
• The cystic fibrosis transmembrane regulator (CFTR) and fibronectin extra domain-A (EDB) are alternatively spliced transcripts with critical functions.

Purpose of the Study:

• To investigate the role of promoter architecture in regulating alternative splicing of CFTR exon 9 and EDB.
• To determine the impact of splicing factor SF2/ASF on promoter-dependent alternative splicing.
• To elucidate the cis-acting elements and kinetic mechanisms involved in promoter-mediated splicing regulation.

Main Methods:

• Hybrid minigene experiments were employed to study alternative splicing.
• Analysis of specific alternative splicing patterns associated with different promoters.
• Investigation of promoter-dependent sensitivity to the splicing factor SF2/ASF.
• Deletion analysis of cis-acting elements within the CFTR minigene.

Main Results:

• Distinct alternative splicing patterns were observed for each analyzed promoter.
• Promoter-dependent regulation by SF2/ASF was specific to CFTR exon 9 and abolished upon deletion of a silencer element.
• CFTR exon 9 sensitivity to SF2/ASF was lost with suboptimal splice sites or deleted exonic enhancers, while basal promoter effects remained.
• EDB splicing was refractory to promoter-mediated regulation by SF2/ASF.

Conclusions:

• Promoter architecture plays a significant role in modulating alternative splicing outcomes.
• The sensitivity of CFTR exon 9 splicing to SF2/ASF is influenced by promoter-specific elements and cis-acting regulatory sequences.
• A kinetic model involving differential RNA polymerase II elongation is proposed to explain promoter-dependent alternative splicing regulation.