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

Abstract

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups, e.g. tunicate chordates, but not in others, e.g. vertebrates. Functional and evolutionary aspects of SL trans-splicing remain poorly understood and although genome-wide characterization of the trans-spliced mRNA subpopulation would give useful insight, this 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 Roche/454 pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ~250,000 high-quality reads corresponding to 8,790 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 over-representation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca2+ homeostasis, and actin cytoskeleton - eukaryotic cell features widely exploited in the metazoa for cell-cell interaction and signaling.