Genome-wide analysis of alternative splicing in Caenorhabditis elegans

Alternative splicing (AS) is the process by which multiple mRNA transcripts are produced from a single precursor transcript through the differential utilization of splice sites. Alternative splicing is one of the key mechanisms that have evolved in metazoans to generate increased transcriptome complexity and recent studies estimate that greater than 95% of human multi-exon genes express multiple splice isoforms (Pan et al. 2008; Wang et al. 2008). Moreover, alternatively spliced exons are often differentially regulated across tissues and during development, suggesting that individual isoforms may serve specific spatial or temporal roles (Hartmann and Valcarcel 2009; Licatalosi and Darnell 2010; Nilsen and Graveley 2010). The importance of proper regulation of AS during development has been demonstrated in many different instances; one particularly well-studied example is that of the sex determination pathway in Drosophila. In this pathway, the female-specific expression of a splicing regulator transformer stimulates the inclusion of exons in transcripts of the doublesex and fruitless transcription factor genes (Lopez 1998; Forch and Valcarcel 2003). The female-specific isoforms of these transcription factors subsequently activate the expression of genes required for female development, while the male-specific variants induce a gene expression program important for male differentiation (Dulac 2005; Shirangi and McKeown 2007). Similar spatio-temporally regulated AS networks are likely to exist in metazoans. The characterization of these AS networks, and their integration with other layers of gene regulation, will be necessary for a more complete understanding of development (Blencowe 2006). The nematode C. elegans, with its amenability for genetic manipulations, defined cell lineage, simple but extensively differentiated nervous system, conserved splicing machinery, and established molecular and cell biological resources, provides an attractive yet relatively under-explored model organism with which to study the regulation and functions of AS in vivo. Tissue or developmental stage-specific AS events have been identified in approximately 70 C. elegans genes which, like those identified in vertebrates, are controlled by combinatorial interactions between splicing factors (Kuroyanagi et al. 2007; Barberan-Soler and Zahler 2008; Ohno et al. 2008). These studies, while informative, were limited to the analysis of isoforms represented in expressed sequence tag (EST)/cDNA libraries, which currently do not provide a full coverage of the transcriptome. The advent of deep sequencing technologies has now enabled transcriptome analyses at unprecedented levels of sensitivity and precision, and these have recently been applied in C. elegans (Hillier et al. 2009; Ramani et al. 2009; Mangone et al. 2010). However, due in part to limited depth of coverage of existing data sets and the ongoing development of analytical software, it has remained a major challenge to systematically categorize and quantify the relative usage of isoforms across development for both known and novel AS events. In this study, we combined high-throughput sequencing of mRNA (RNA-seq) and quantitative AS microarray profiling to provide a high resolution survey of splicing across C. elegans development. We have identified thousands of new AS events and hundreds of isoforms that are differentially expressed during development. Additionally, we identified candidate cis-elements that likely play a role in regulating these events. To facilitate the use of these data, we have created the “C. elegans Splice Browser,” a web resource in which all of the data in the present study are made accessible. This resource, which represents the most complete set of splice variants reported to date, should serve as a valuable basis for focused analyses of the functional importance of individual splice isoforms in vivo.