Regulatory Elements and Splicing Factors Govern Mutually Exclusive Splicing of DmRdl Exon 3

Ionotropic γ-aminobutyric acid (GABA) receptors are major inhibitory receptors in insect central nervous systems, with their diversity partly generated by alternative splicing. In Drosophila melanogaster, mutually exclusive alternative splicing (MEAS) of the resistant to dieldrin gene (Rdl) exon 3 (E3) produces Rdl-3a and Rdl-3b, but its regulatory mechanism remains unclear. In the present study, two intronic regulatory elements (IREs) and one exonic regulatory element (ERE) were identified in the exon 2 (E2)–exon 4 (E4) region of DmRdl precursor mRNA (pre-mRNA). The IRE1 mutation decreased exon 3b (E3b) inclusion from 54.12% to 3.95%, whereas the IRE2 mutation increased E3b inclusion from 61.70% to 97.82%. The ERE mutation significantly increased E3b inclusion both in vitro and in vivo, while the IRE mutations were lethal in vivo. The results of RNA pull-down, mass spectrometry, and RNAi demonstrated that the splicing factors, CG30122 and Rbp1-like bind to the ERE and exert opposite effects on E3b inclusion. The results of STRING network analysis supported the functional interaction between CG30122 and Rbp1-like, while molecular docking provided structural support for their potential interaction with the ERE-containing RNA sequence. This study reveals the molecular regulatory network underlying the MEAS of DmRdl E3, and and provides a foundation for investigating other MEAS exons, such as E6 and E9 in insect Rdl.