Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.

Area of Science:

• Entomology
• Molecular Biology
• Genetics

Background:

• Doublesex (dsx) is a crucial regulator of insect sex determination.
• While well-studied in Drosophila, the sex determination pathway in Lepidoptera remains less understood.
• This study focuses on the alternative splicing of the dsx gene in key Lepidopteran species.

Purpose of the Study:

• To investigate the alternative splicing of the doublesex (dsx) gene in Lepidoptera.
• To identify conserved RNA elements and regulatory mechanisms involved in sex-specific dsx splicing.
• To understand the functional role of these elements in female-specific dsx mRNA production.

Main Methods:

• Comparative analysis of dsx mRNA and protein sequences across Lepidopteran species.
• Splice site strength analysis and experimental confirmation.
• Identification and characterization of conserved RNA cis-elements.
• Mutagenesis studies using mini-genes and cell line expression.

Main Results:

• Discovery of over a dozen novel alternative splicing isoforms of dsx in female Lepidoptera.
• Identification of highly conserved RNA elements (I, II, III, IV, 3E-1, 3E-2) in Lepidopteran dsx.
• Experimental validation of a weak 5' splice site at intron 3.
• Demonstration that RNA elements 3E-1 and 3E-2 act as exonic splicing enhancers for female-specific splicing.

Conclusions:

• The weak 5' splice site at intron 3 in Lepidopteran dsx is critical for alternative splicing.
• Conserved exonic splicing enhancers (3E-1, 3E-2) facilitate female-specific dsx mRNA production.
• These findings elucidate a novel mechanism for sex determination in Lepidoptera.