Resolution of polycistronic RNA by SL2 -splicing is a widely conserved nematode trait.

Spliced leader -splicing is essential for the processing and translation of polycistronic RNAs generated by eukaryotic operons. In , a specialized spliced leader, SL2, provides the 5' end for uncapped pre-mRNAs derived from polycistronic RNAs. Studies of other nematodes suggested that SL2-type -splicing is a relatively recent innovation, confined to Rhabditina, the clade containing and its close relatives. Here we conduct a survey of transcriptome-wide spliced leader -splicing in , a distant relative of with a particularly diverse repertoire of 15 spliced leaders. By systematically comparing the genomic context of -splicing events for each spliced leader, we identified a subset of spliced leaders that are specifically used to process polycistronic RNAs-the first examples of SL2-type spliced leaders outside of Rhabditina. These spliced leader RNAs possess a perfectly conserved stem-loop motif previously shown to be essential for SL2-type -splicing in We show that genes -spliced to these SL2-type spliced leaders are organized in operonic fashion, with short intercistronic distances. A subset of operons show conservation of synteny with operons. Our work substantially revises our understanding of nematode spliced leader -splicing, showing that SL2 -splicing is a major mechanism for nematode polycistronic RNA processing, which may have evolved prior to the radiation of the Nematoda. This work has important implications for the improvement of genome annotation pipelines in nematodes and other eukaryotes with operonic gene organization.