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

In contrast to canonical cis-splicing, trans-splicing combines exons from two distinct transcripts, yielding chimeric mRNAs. One striking example is the ubiquitously expressed modifier of the mdg4 (mod(mdg4)) locus in Drosophila, where all mRNAs spanning over 30 isoforms are generated exclusively by trans-splicing. This process combines common constitutive exons with one of many alternative 3' variable exons transcribed from independent promoters. Here, we demonstrate that trans-splicing is determined by a 600-bp region located in the proximal part of the last common intron. Unlike other known examples of trans-splicing, complementary sequences between donor and acceptor pre-mRNAs do not trigger trans-splicing of the mod(mdg4) gene. Deletion analysis revealed that the 600-bp intronic region contains multiple redundant motifs for RNA-binding proteins (RBPs) that collectively determine the level of trans-splicing. We identified specific binding of a large group of spliceosome-associated RBPs by affinity purification of the in vitro transcribed proximal part of the intronic RNA. A multifold reduction in mRNA expression caused by the partial removal of intronic sequences is partially compensated for by an increase in the translation efficiency of Mod(mdg4) isoforms. The results were consistent with a model in which multiple RBP motifs stabilize pre-mRNA binding to the spliceosome, thereby inducing trans-splicing.