Intron loss is associated with gain of function in the evolution of the gloverin family of antibacterial genes in Bombyx mori.

Gene duplication is a characteristic feature of eukaryotic genomes. Here we investigated the role of gene duplication in the evolution of the gloverin family of antibacterial genes (Bmglv1, Bmglv2, Bmglv3, and Bmglv4) in Bombyx mori. We observed the following two significant changes during the first duplication event: (i) loss of intronV, located in the 3'-untranslated region (UTR) of the ancestral gene Bmglv1, and (ii) 12-bp deletion in exon3. We show that loss of intronV during Bmglv1 to Bmglv2 duplication was associated with embryonic expression of Bmglv2. Gel mobility shift, chromatin immunoprecipitation, and immunodepletion assays identified chorion factor 2, a zinc finger protein, as the repressor molecule that bound to a 10-bp regulatory motif in intronV of Bmglv1 and repressed its transcription. gloverin paralogs that lacked intronV were independent of chorion factor 2 regulation and expressed in embryo. These results suggest that change in cis-regulation because of intron loss resulted in embryonic expression of Bmglv2-4, a gain of function over Bmglv1. Studies on the significance of intron loss have focused on introns present within the coding sequences for their potential effect on the open reading frame, whereas introns present in the UTRs of the genes were not given due attention. This study emphasizes the regulatory function of the 3'-UTR intron. In addition, we also studied the genomic loss and show that "in-frame" deletion of 12 nucleotides led to loss of amino acids IHDF resulting in the generation of a prepro-processing site in BmGlv2. As a result, the N-terminal pro-part of BmGlv2, but not of BmGlv1, gets processed in an infection-dependent manner suggesting that prepro-processing is an evolved feature in Gloverins.