Structural basis of step II spliced leader RNA trans-splicing in trypanosomatid parasites

Messenger RNA (mRNA) synthesis is fundamental to eukaryotic gene expression. In kinetoplastid parasites, including the human pathogens Trypanosoma and Leishmania, mRNAs are produced through a mechanism called Spliced Leader (SL) RNA trans-splicing. In this process, a short SL exon from a small noncoding RNA is joined to the 5’ end of every mRNA, ensuring transcript stability and translation. Despite decades of study, the structural and mechanistic basis of SL trans-splicing remains elusive. Here, we report cryogenic electron microscopy structures of step II SL trans-splicing machineries from Leishmania tarentolae. These structures reveal the molecular mechanism of SL exon ligation and uncover lineage-specific adaptations that remodel the trypanosomatid trans-spliceosome for SL trans-splicing while preserving fundamental spliceosomal chemistry. Altogether, our results establish a mechanistic framework for SL trans-splicing and illuminate the evolutionary diversification of RNA processing in deeply diverged eukaryotes.