Direct repression of splicing by transformer-2.

Area of Science:

• Molecular Biology
• Genetics
• RNA Splicing Regulation

Background:

• Transformer-2 (Tra2) is a key regulator in Drosophila melanogaster sex determination.
• Tra2 positively influences doublesex (dsx) and fruitless (fru) pre-mRNA splicing.
• A negative-feedback loop exists where Tra2 limits its own synthesis via M1 intron retention in its pre-mRNA, but the mechanism was unclear.

Purpose of the Study:

• To elucidate the molecular mechanism by which Tra2 negatively regulates its own pre-mRNA splicing.
• To identify the specific sequences and protein interactions involved in M1 intron retention.

Main Methods:

• In vitro splicing assays using recombinant Tra2 protein and Drosophila nuclear extracts.
• RNA-binding studies to map Tra2 interaction sites on tra2 pre-mRNA.
• Functional analysis of RNA sequences critical for M1 intron repression and splicing enhancement.

Main Results:

• Recombinant Tra2 protein directly represses M1 intron splicing in vitro.
• Tra2 binds to multiple sites within and near the M1 intron, including an A/C-rich sequence upstream of the 5' splice site.
• Competition for Tra2 binding restores M1 splicing, and the A/C-rich element enhances M1 splicing in the absence of Tra2.

Conclusions:

• Tra2 protein directly represses M1 intron splicing through binding to specific regulatory elements.
• The A/C-rich sequence, also found in enhancer elements of dsx and fru, is crucial for Tra2-mediated repression.
• Tra2 likely interferes with enhancer function to inhibit M1 splicing, completing its negative-feedback loop.