High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: Alternative expression modes and gene function correlates

Pre-mRNA 5′ spliced-leader (SL) trans -splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans -spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans -spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ∼250,000 high-quality reads corresponding to 8790 genes, ∼58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans -splicing, including the existence of a significant class of “infrequently trans -spliced” genes, accounting for ∼28% of represented genes, that generate largely non- trans -spliced mRNAs, but also produce trans -spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans -spliced versus non- trans -spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans -spliced gene categories. Our data include reads representing ∼80% of Ciona frequently trans -spliced genes. Our analysis also revealed significant use of closely spaced alternative trans -splice acceptor sites which further underscores the mechanistic similarity of cis - and trans -splicing and indicates that the prevalence of ±3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans -splicing status and gene function, namely the overrepresentation in the frequently trans -spliced gene class of genes associated with plasma/endomembrane system, Ca 2+ homeostasis, and actin cytoskeleton.