Alternative splicing is a powerful mechanism by which a single gene can give rise to multiple mRNA isoforms, and therefore multiple proteins. Because different protein isoforms can have distinct biological functions, alternative splicing plays an important role in regulating gene expression [4
]. Alternative splicing also plays a central role in expanding the size and diversity of the proteome. In fact, it is estimated that as many as 74% of human genes encode alternatively spliced transcripts [5
]. Despite the prevalence of alternative splicing, we have a very limited knowledge about the underlying regulatory mechanisms. For example, only a handful of splicing regulators has been identified to date and the endogenous regulatory targets for most of these regulators are not known. Given the sheer number of alternatively spliced exons that exists in metazoan organisms, it is almost certain that numerous splicing regulatory factors have yet to be discovered.
To begin identifying proteins involved in regulating alternative splicing, we have performed an RNA interference (RNAi) screen in cultured Drosophila melanogaster
cells. D. melanogaster
is an excellent model organism to study alternative splicing for several reasons. First, the components of the spliceosome are highly conserved between human and the fruitfly [8
]. Second, it is easy to perform both genetic and biochemical experiments using the animals and extracts from cultured cells or embryos, respectively. Finally, it is relatively easy and inexpensive to perform RNAi in Drosophila
cells. In contrast to mammalian cells, where expensive synthetic siRNAs or vectors that express shRNAs are used, RNAi in cultured Drosophila
cells can be performed by simply adding long (~400–500 bps) dsRNAs that can be enzymatically synthesized in the lab to the culture media [10
]. The simplicity of this system has allowed several genome-wide RNAi screens to be performed in Drosophila
culture cells and have led to the identification factors involved in biological processes ranging from cell viability to cell morphology [6
]. In this chapter, we will describe in detail the RNAi approach we use to identify and analyze RNA binding proteins involved in controlling alternative splicing.