The pheromone genes of the self/non-self recognition mechanism of the ciliate Euplotes crassus generate multiple transcripts by an alternative splicing of ‘matryoshka’ introns

In ciliates, cell-type distinctive protein pheromones control a self/non-self recognition mechanism responsible for the cell switching between the vegetative and sexual stages of the life cycle. These signaling molecules are encoded by genes (pheromone genes) that in the cell somatic nucleus (macronucleus) represent the transcriptionally active versions of transcriptionally silent genes allelic at the same genetic locus mat of the cell germinal nucleus (micronucleus). In the course of evolution, in Euplotes, the native single multiallelic mat locus underwent duplication among species which, such as E. crassus, form the latest branching clade of the phylogenetic tree. Because of this duplication, E. crassus expresses two distinct families of pheromone genes instead of a single family, as is the case in species of earlier branching clades. We analyzed the structure and expression of a number of E. crassus pheromone genes representative of the two families. Like their orthologs of other Euplotes species, these genes show 5’-leader regions that are much more extended than the coding regions, lack canonical regulatory sequences for gene transcription, and synthesize multiple transcripts (in addition to the pheromone-specific one) through the activity of two distinct transcription start sites and a mechanism of alternative intron splicing. These E. crassus introns have been found to be unique with respect to introns of all the other Euplotes pheromone genes. They can be distinguished between ‘matryoshka’ introns, residing one inside the other like Russian Dolls, and ‘non-matryoshka’ introns. While the former possess canonical GTA/TAG splicing sites, the latter possess CTA/TAC splicing sites complementary to the canonical GTA/TAG splicing sites. This strongly suggests that both the DNA strands of the E. crassus pheromone genes can be used as template for transcription. We are currently attempting to verify this hypothesis and assign a function to the products of the multiple E. crassus pheromone gene transcripts.