Multiple intron motifs have redundant functions in trans-splicing of the mod(mdg4) locus

In contrast to canonical cis-splicing, trans-splicing combines exons from two distinct transcripts, yielding chimeric mRNAs. This process is typically suppressed through the tight coupling of pre-mRNA synthesis and splicing, primarily due to the inherent risk of generating chimeric proteins with aberrant or deleterious properties. Nevertheless, trans-splicing permits the creation of a substantially greater diversity of mRNA isoforms that encode variants of a single protein. One compelling example is the mod(mdg4) locus in Drosophila, where all mRNAs, encompassing over 30 isoforms, are exclusively generated via trans-splicing, which integrates common constitutive exons with one of several alternative 3′ variable exons transcribed from independent promoters. This investigation analyzed the roles of the mod(mdg4) gene’s promoter, exons, and introns in trans-splicing. Our methodology involved a previously validated model in the heterogeneous 22A genomic region, augmented by targeted deletions within the fourth intron of the endogenous locus. We determined that the mod(mdg4) promoter only modestly enhances trans-splicing efficiency. Critically, the predominant determinants are multiple, redundant RNA-binding protein motifs concentrated within the proximal 412 bp of the fourth intron. These results collectively support a model wherein trans-splicing is primarily mediated by redundant intronic motifs recognized by spliceosome components. RNA-seq experiments were performed on whole 2–3 day-old adult male Drosophila from the wild-type control line y[1]w[1118] and the mod(mdg4)[c13] mutant line, in which an intronic fragment spanning positions 225–553 was deleted and only the c13 motif was integrated. Total RNA was extracted and enriched by hybridization with a biotinylated probe targeting constitutive exons to increase the proportion of reads mapping to the locus of interest. Changes in trans-splicing patterns across all isoforms were compared between the control and mutant lines.