The organisation of genes into operons - clusters of genes which are transcribed as polycistronic RNAs - is a feature of all known nematode genomes. Spliced leader trans-splicing is essential for the expression of downstream operonic genes because the spliced leader provides the 5' cap to their otherwise uncapped transcripts. In C. elegans, a specialised spliced leader, SL2, is specific for transcripts derived from downstream operonic genes via a process that is mechanistically distinct from the more generic SL1 trans-splicing. Studies of nematodes outside of Clade V failed to detect SL2 trans-splicing, with downstream operonic gene transcripts being trans-spliced to SL1. This led to the hypothesis that SL2 trans-splicing is recent innovation and that SL1 trans-splicing is the ancestral mechanism for resolving nematode polycistronic RNAs. However, a rigorous investigation of this hypothesis requires the comprehensive genome-wide characterisation of both operons and spliced leader trans-splicing. This has, until recently, been challenging, and their identification has historically relied upon sequence similarity with C. elegans, which may bias the results.To systematically investigate spliced leader trans-splicing and operon organisation, we have developed two fully automated discovery and annotation pipelines, SLIDR and SLOPPR (https://doi.org/10.1186/s12859-021-04009-7), that enable the comprehensive characterisation of spliced leader trans-splicing and operon organisation in any organism using standard RNA-Seq datasets. Using these tools, we showed that SL2 trans-splicing is more broadly distributed than previous studies suggested; it is found in all Clade I nematodes that we investigated
(http://www.rnajournal.org/cgi/doi/10.1261/rna.076414.120). However, we were unable to detect SL2 trans-splicing in any Clade III nematode, consistent with previous studies, and could only detect it in a small sub-set of Clade IV nematodes. These distributions can be explained either by loss of an ancestral SL2 trans-splicing mechanism in multiple lineages, with SL1 acquiring the role in processing polycistronic RNA; or by the convergent evolution of SL2 trans-splicing in selected lineages. I will present data that favours the former explanation provides a possible scenario to explain how SL1 might replace SL2 trans-splicing despite the latter's broad conservation and therefore functional importance.