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.

Background/Objectives: Glutamate-gated chloride channels (GluCls) are essential inhibitory neurotransmitter receptors in insects and key targets of several insecticides. In Spodoptera frugiperda (FAW), mutually exclusive alternative splicing (MEAS) of exon 3 in SfGluCl produces three splice variants (SfGluCl 3A, SfGluCl 3B and SfGluCl 3C), yet the cis-regulatory elements involved in this splicing event remain unknown. This study aimed to identify candidate cis-regulatory regions associated with exon 3 MEAS and characterize their effects on exon 3 selection in a Sf9 cell-based minigene system. Methods: Semi-quantitative RT-PCR was used to assess the expression levels of three splice variants of exon 3 in SfGluCl across developmental stages. Comparative sequence analysis, combined with intron-deletion and exon-exchange assays, were performed to identify candidate cis-regulatory regions. A total of 27 constructs (23 intron-deletion and 4 exon-exchange constructs) were generated and analyzed using a minigene splicing assay in Sf9 cells. Results: Three splice variants were consistently detected across all developmental stages. In the Sf9 cell-based minigene system, two candidate cis-regulatory regions affecting SfGluCl 3B inclusion were identified: a 20 bp intronic region spanning nucleotides +13 to +32 downstream of the 5′ splice junction in the intron between exons 3B and 3C, corresponding to the region deleted in ExonB-C (Exon 3B-exon 3C)-Intdel52, and an 11 bp region within exon 3B containing six nucleotide substitutions in Ex3B (Exchanged exon 3B)-SubA2. Deleting the ExonB-C-Intdel52 or modifying the Ex3B-SubA2 significantly increased the SfGluCl 3B inclusion. Conclusions: These candidate intronic and exonic regions can influence exon 3B inclusion in the Sf9 cell-based minigene system. Although their functional effects and in vivo relevance remain to be validated in further study, our findings still provide a basis for future studies of GluCl splice variant regulation, receptor biology, and insecticide response in insects.