Unexpected intron plasticity and trans-splicing capability suggest spliceosome diversification in the evolutionarily...

Spliceosomal introns, distinctive features of eukaryotic genomes, are non-coding sequences excised from pre-mRNAs by the spliceosome, contributing to genome evolution and protein diversity. Although spliceosomal introns have been characterised in several eukaryotic lineages, their origin and evolution remain unresolved. The protozoan parasite Trichomonas vaginalis is a highly divergent eukaryote with a large genome and a rich gene repertoire, but apparently few spliceosomal introns. Following from the discovery of a group of unusually short introns in this organism, we developed here a fluorescent reporter system and combined with extensive mutagenesis to dissect the splicing requirements of these short introns, comparing them to conventional long introns. We found that short introns have reasonable but limited flexibility in their length, including extreme juxtaposition of the branch site and the 3' splice site, which, to our knowledge, is an unprecedented feature among eukaryotic introns. Additionally, they clearly exhibit splice signal features that distinguish them from long introns, including a highly degenerate 5' splice site. Remarkably, we found that T. vaginalis is capable of trans-splicing an endogenous intron that was deliberately split and a naturally split intron from Giardia lamblia, a more distant diplomonad within the same Metamonada supergroup. Collectively, our findings highlight the evolutionary plasticity of RNA splicing systems in divergent eukaryotes, offering new perspectives on splicing mechanisms by the spliceosome.