The Exon Junction Complex and Srp54 contribute to Drosophila Hedgehog signaling via ci RNA splicing

Hedgehog (Hh) regulates the Cubitus interruptus (Ci) transcription factor in Drosophila melanogaster by activating full-length Ci-155 and blocking processing to Ci-75 repressor. However, the interplay between regulation of Ci-155 levels and activity, as well as processing-independent mechanisms that affect Ci-155 levels have not been explored extensively. Here we identified Mago Nashi (Mago) and Y14 core Exon Junction Complex (EJC) proteins, as well as the Srp54 splicing factor as modifiers of Hh pathway activity under sensitized conditions. Mago inhibition reduced Hh pathway activity by altering the splicing pattern of ci to reduce Ci-155 levels. Srp54 inhibition also affected pathway activity by reducing ci RNA levels but additionally altered Ci-155 levels and activity independently of ci splicing. Further tests using ci transgenes and ci mutations confirmed evidence from studying the effects of Mago and Srp54 that relatively small changes in the level of Ci-155 primary translation product alter Hh pathway activity under a variety of sensitized conditions. We additionally used ci transgenes lacking intron sequences or the presumed translation initiation codon for an alternatively spliced ci RNA to provide further evidence that Mago acts principally by modulating the levels of the major ci RNA encoding Ci-155, and to show that ci introns are necessary to support production of sufficient Ci-155 for robust Hh signaling and may also be important mediators of regulatory inputs.