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

Abstract

Pre-mRNA 59 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 highthroughput 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 63-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.[Supplemental material is available online at http://www.genome.org. The sequence data from this study have been submitted to the NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession no. SRX006190.]A striking evolutionary variation in eukaryotic gene expression mechanisms is the presence or absence in diverse organismal groups of a form of RNA splicing-pre-mRNA spliced leader (SL) trans-splicing (Davis 1996;Nilsen 2001;Hastings 2005). SL transsplicing is closely related to conventional RNA splicing, or cissplicing. The same nucleotide sequence features define donor and acceptor sites, and both processes occur in spliceosomes and involve formation of a 59,39,29 branchpoint upstream of the acceptor site (Agabian 1990;Nilsen 1993). Whereas cis-splicing joins paired donor and acceptor sites within a single RNA molecule, in SL transsplicing the donor exon is the 59-segment of a specialized small 59-capped RNA molecule-the SL RNA-and the target is an unpaired acceptor site near the 59-end of a pre-mRNA molecule.Transfer of the SL RNA 59-segment, the SL sequence, to the pre-mRNA molecule occurs with loss of the pre-mRNA's initial 59-segment ups...

SL2-like spliced leader RNAs in the basal nematode Prionchulus punctatus: New insight into the evolution of nematode SL2 RNAs

“…The range of organisms in which SL trans-splicing has been identified is broad, extending from protists through to chordates (Sutton and Boothroyd 1986;Tessier et al 1991;Davis 1997;Stover and Steele 2001;Blumenthal 2005;Pouchkina-Stantcheva and Tunnacliffe 2005;Zayas et al 2005;Guiliano and Blaxter 2006;Zhang et al 2007;Marletaz et al 2008;Pettitt et al 2008;Zhang and Lin 2009;Derelle et al 2010;Douris et al 2010;Matsumoto et al 2010), although it appears to be absent from plants, fungi, and vertebrates. The phylogenetic distribution of SL transsplicing is sporadic, however, and does not allow one to distinguish whether this distribution reflects a single origin 1 These authors contributed equally to this work.…”

Trans-splicing in metazoans: A link to translational control?

“…Trans-spliced mRNAs in C. intestinalis do not include TOP mRNAs but instead are enriched for genes related to plasma and endomembrane systems, Ca 2C homeostasis/regulation, cell-cell signaling and cortical cytoskeleton. 3 These are all related to important processes in the maturation of oocytes: the cytoskeleton is critical in the reorganization of organelles; Ca 2C signaling pathways are remodeled, with accompanying changes in membrane permeability and ion currents, in preparation for fertilization. Further studies of translational control in these and other species that utilize trans-splicing are needed to increase our understanding of this phenomenon and the forces that drive its appearance in various, disparately related, lineages.…”

“…In the ascidian C. intestinalis, 58% of mRNAs are trans-spliced with a single SL species and 20% of genes are organized into operons. 3 In the appendicularian Oikopleura dioica, 39% of mRNAs are trans-spliced to a single SL and 28% are found in operons. [4][5][6] Several hypotheses for the functions and evolutionary advantages of both operons and trans-splicing ( Fig.…”

Evolutionary crossroads in developmental biology: the tunicates

“…These genomes are surprisingly small (Ciona~160 Mb; Oikopleura~70 Mb, the smallest animal genome so far), owing to several factors: their unduplicated gene complement; substantial gene loss (Holland and Gibson (Satou et al, 2008;Denoeud et al, 2010;Ganot et al, 2004). Frequent or infrequent trans-splicing is also found in most monocistronic genes (Matsumoto et al, 2010).…”

“…These genomes are surprisingly small (Ciona~160 Mb; Oikopleura~70 Mb, the smallest animal genome so far), owing to several factors: their unduplicated gene complement; substantial gene loss (Holland and Gibson (Satou et al, 2008;Denoeud et al, 2010;Ganot et al, 2004). Frequent or infrequent trans-splicing is also found in most monocistronic genes (Matsumoto et al, 2010).Tunicate genomes evolve rapidly. Some tunicate proteins have among the fastest evolution rates of metazoans (Denoeud et al, 2010;Putnam et al, 2008), and non-coding cis-regulatory elements of phylogenetically distant ascidians, such as Ciona and Halocynthia, can diverge up to a point at which their sequences cannot be aligned (Oda-Ishii et al, 2005).…”