Surprising diversity and distribution of spliced leader RNAs in flatworms

Trans-splicing generates the mature 5' ends of certain mRNAs through the addition of a small spliced leader (SL) exon to pre-mRNAs. To search for novel flatworm spliced leaders, degenerate oligonucleotides and 5' RACE [corrected was used to isolate and characterize the 5' terminal sequences of enolase mRNAs in diverse flatworms. Several new spliced leaders and their SL RNA genes were identified, characterized, and compared. All parasitic trematodes examined trans-splice enolase. A primitive polyclad turbellarian, Stylochus zebra, also contains a trans-spliced enolase mRNA. The S. zebra SL is the longest SL yet identified, 51 nucleotides. Comparison of flatworm SLs indicates that they vary significantly in sequence and length. This suggests that neither spliced leader exon sequence nor size is likely to be essential for trans-splicing in flatworms. Flatworm SL RNAs have unusual Sm binding sites with characteristics distinct from other known flatworm snRNA Sm binding sites. Predicted flatworm SL RNA secondary structures show variation exhibiting 2-4 stem loops. Although limited in sequence similarity, phylogenetically conserved regions within the diverse flatworm SL RNAs suggest that they are likely to be derived from a common ancestor and provide information on potentially important SL RNA elements. The identification of a SL in a primitive flatworm suggests that trans-splicing may have been an ancestral feature in the phylum. Representative species of other early and more recent clades within the phylum, however, do not trans-splice enolase, nor do they or representatives of several other flatworm groups, have an SL RNA with a phylogenetically conserved region identified in the current study.