Phylogenetic Analysis of Step II Trans-Spliceosome Factors across Discoba

Abstract

Kinetoplastids are a group of flagellated protists that include the genera Leishmania and Trypanosoma, which are responsible for several Neglected Tropical Diseases affecting millions of people worldwide. These organisms possess highly divergent molecular mechanisms, including spliced leader (SL) trans-splicing, an essential RNA-processing pathway in which polycistronic precursor RNAs are processed into individual mature mRNAs and fused to a conserved 39-nucleotide spliced leader exon at their 5′ end. Despite its fundamental role in gene expression, the molecular basis and evolutionary origin of this process remain incompletely understood. Recent cryo-electron microscopy (cryo-EM) structures of the Leishmania tarentolae step II trans-spliceosome have revealed numerous previously uncharacterized trans-splicing factors. To investigate the evolutionary history of these factors and identify their orthologs in other medically relevant kinetoplastids and related species within the Discoba clade, we performed a comprehensive phylogenomic analysis. Datasets of newly identified trans-splicing factors, together with conserved cis-splicing factors, were assembled from available proteomic and genomic resources across Discoba. Individual gene phylogenies and a species tree were then reconstructed to infer the taxonomic distribution and evolutionary trajectories of these proteins. Overall, this work provides a comprehensive evolutionary framework for trans-splicing factors and offers new insights into the evolution of RNA splicing by enabling the near-complete identification of step II trans-spliceosome components and related factors across this highly divergent and medically important eukaryotic lineage.