Genome-wide analysis of trans-splicing in the nematode Pristionchus pacificus unravels conserved gene functions for germline and dauer development in divergent operons

Discovery of trans-splicingin multiple metazoan lineages led to the identification of operon-like gene organization in diverse organisms, including trypanosomes, tunicates, and nematodes, but the functional significance of such operonsis not completely understood. To see whether the content or organization of operonsserves similar roles across species, we experimentally defined operonsin the nematode model Pristionchus pacificus. We performed affinity capture experiments on mRNA pools to specifically enrich for transcripts that are trans-splicedto either the SL1- or SL2-spliced leader, using spliced leader-specific probes. We obtained distinct trans-splicingpatterns from the analysis of three mRNA pools (total mRNA, SL1 and SL2 fraction) by RNA-seq. This information was combined with a genome-wide analysis of gene orientation and spacing. We could confirm 2219 operonsby RNA-seqdata out of 6709 candidate operons, which were predicted by sequence information alone. Our gene ordercomparison of the Caenorhabditis elegans and P. pacificus genomes shows major changes in operonorganization in the two species. Notably, only 128 out of 1288 operonsin C. elegans are conserved in P. pacificus. However, analysis of gene-expression profiles identified conserved functions such as an enrichment of germline-expressed genes and higher expression levels of operonicgenes during recovery from dauer arrest in both species. These results provide support for the model that a necessity for increased transcriptional efficiency in the context of certain developmental processes could be a selective constraint for operonevolution in metazoans. Our method is generally applicable to other metazoans to see if similar functional constraints regulate gene organization into operons.