The search for alternative splicing regulators: new approaches offer a path to a splicing code

Abstract

Complex multicellular organisms require a diverse set of proteins to set the form and function of specialized cell types. The availability of complete genomic sequences has revealed that instead of a large increase in the number of protein coding genes compared with unicellular organisms, more complex eukaryotes instead obtain more diversity out of a relatively limited number of genes through the process of alternative splicing (AS). AS results in the cell type-, developmental stage-, sex-, or signal-regulated changes in composition of an mRNA produced from a given gene, brought about by changes in splice site choice (Black 2003; Matlin et al. 2005). There are many different types of AS events, ranging from the tissue-specific inclusion of a cassette exon to the Dscam gene in Drosophila, which contains four clusters of exons containing 12, 48, 33, and 2 mutually exclusive variants, an extreme example of AS complexity (Bharadwaj and Kolodkin 2006). Additionally, it is clear from the relatively small number of AS events that have been studied extensively at a mechanistic level that regulation of AS takes many forms, as will be discussed in more detail later in this article. Lastly, the functional outcomes of AS vary greatly, from effectively turning off a gene (the result of including an exon containing a premature stop codon, for example), to a subtle change in a protein’s function. In spite of this complexity, the goal of understanding the changes in AS patterns in terms of changes in expression and regulation of factors that regulate AS across cell types appears achievable. This is in part because recent technical advances allow us, starting with an individual splicing factor, to determine its genome-wide role in AS regulation. However, the ambitious goal of determining a cellular code for AS will be impossible to realize without a complete list of AS regulators, and currently there is no reason to believe we are anywhere near completing such a list in any metazoan. Kuroyanagi et al. (2006) have developed recently a system in Caenorhabditis elegans, which allows for the straightforward visualization of individual AS events in vivo, which in turn allows for the identification of mutants defective in regulating the event. Using this approach, Kuroyanagi and colleagues (Kuroyanagi et al. 2006, 2007; Ohno et al. 2008) have identified four proteins that regulate two different AS events, a feat that would be laborious using in vitro approaches. Three of these proteins had not been shown previously to participate in regulation of AS, indicating that we may be far from a complete list of AS regulators. Their data also highlight the fact that regulators of AS are often highly conserved throughout metazoans, meaning that filling the list of splicing regulators in C. elegans will likely contribute to filling our own.