Introduction

The majority of metazoan protein-coding genes contain intervening sequences (introns) whose removal from precursor messenger RNAs (pre-mRNAs) is an essential step in gene expression. The excision of these noncoding sequences (i.e., introns) and joining of coding sequences (exons) is catalyzed by a large macromolecular complex called the spliceosome, which is composed of five small nuclear RNAs (U1, U2, U4, U5, and U6) and numerous small nuclear ribonucleo-protein (snRNP) and non-snRNP proteins (1,2). Three cis acting elements within the pre-mRNA, known as the 5′ and 3′ splice sites and the branchpoint sequence (BPS) direct the assembly of the spliceosome. Thus, the spliceosome performs two functions: recognition of the intron/exon boundaries and catalysis of the intron removal with simultaneous joining of the exons.

Pre-mRNAs can also undergo alternative splicing, a precisely regulated process in which differential joining of 5′ and 3′ splice sites of a single pre-mRNA generates variant mRNAs with diverse, and often antagonistic, functions (3,4). Remarkably, the Drosophila Down syndrome cell adhesion molecule (Dscam) gene can potentially generate more than 38,000 isoforms by alternative splicing (5,6,7). There is growing evidence that both extra- and intracellular signaling regulate alternative splicing, which can lead to the skipping or inclusion of exons, retention of introns, shortening or lengthening of exons, and in some cases, inclusions of exons in the mRNA in a mutually exclusive manner (8,9,10,11). It has been estimated that 35%–70% of human genes generate transcripts that are alternatively spliced (12). In addition, 70%–90% of alternative splicing events result in the generation of proteins with diverse functions ranging from sex determination to apoptosis (3)(13,14,15). Alternative splicing of pre-mRNA is now recognized as the most important source of protein diversity in vertebrates (16,17,18).

In general, the splicing signals of alternatively spliced exons do not match with the consensus sequence (19,20). Although the underlying mechanisms that regulate alternative splicing are poorly understood, it has become increasingly clear that the auxiliary cis acting elements, known as exonic and intronic splicing enhancers (ESEs and ISEs, respectively) and exonic and intronic splicing silencers (ESSs and ISSs, respectively), play an important role in the recognition of exons surrounding the regulated splice sites (21). The function of auxiliary cis acting elements is to provide the binding surface for splicing regulators, which in cooperation with other factors communicate with the basal splicing machinery to enhance or inhibit splice-site selection (21,22) (Figure 1). The most common splicing regulators are RNA binding proteins that are members of heterogeneous nuclear ribonucleo-protein (hnRNP) (23,24) and serine/ arginine-rich (SR) protein families (25,26). Thus, regulation of alternative splicing is a combinatorial phenomenon in which cooperative assembly of the activation and/or repression complexes near the regulated exons determine whether a particular exon will be included in the mRNA.

Figure 1. Networks of RNA-RNA, RNA-protein, and protein-protein interactions regulate alternative splice site choice. (Click to enlarge)

Aberrant Splicing and Human Diseases

The defective regulation of splice variant expression has been identified as the cause of several genetic disorders (27,28,29,30,31,32,33), and certain forms of cancer have been linked to unbalanced isoform expression of genes involved in processes ranging from cell cycle regulation to angiogenesis (34,35,36,37,38,39). In general, aberrant splicing is caused by mutations of the cis and trans acting elements. Whereas the mutations of the splice sites, the branchpoint, and auxiliary elements are categorized as cis mutations, trans acting mutations affect the components of the basal splicing machinery or proteins that regulate alternative splicing. Given that aberrant splicing is linked to numerous diseases, the emergence of new technologies for controlling mRNA splicing is not surprising (40,41,42). Importantly, tools that specifically destroy a disease-linked mRNA isoform will have far-reaching effects in medicine and biotechnology.

Emergence of RNAi as a Potential Therapeutic Tool

RNA interference (RNAi) is a remarkable phenomenon by which eukaryotes modulate their gene expression at pre- and posttran-scriptional levels (43,44,45). Although there were some indications of the existence of RNAi in plants (46), the real breakthrough, that RNAi-mediated gene silencing is induced by double-stranded RNA (dsRNA), was demonstrated for the first time in the nematode Caenorhabditis elegans, in which worms injected with dsRNAs could silence homologous genes (47). However, dsRNAs induce immune responses that act as a defense mechanism against viral infection in mammalian cells, which presented a major roadblock to the use of dsRNA for gene silencing (48). To overcome this problem, Tuschl and colleagues designed a novel approach whereby transfection of mammalian cells with chemically synthesized short 21- to 22-nucleotide (nt) RNAs with 2-nt 3’ overhangs could induce sequence-specific gene silencing without nonspecific inhibition of translation (49). This elegant work combined with the discovery that microRNAs (miRNAs) (50,51,52) act as endogenous triggers of the RNAi laid the foundation for in vivo expression of siRNAs using short hairpin RNAs (shRNAs) as mimics of miRNAs (53,54,55,56,57,58). The shRNAs varying in size and length of the stem can be expressed using Pol III promoter, such as U6, H1, and transfer RNA (tRNA) promoters. Alternatively, the Pol III system can be designed to express the sense and the antisense strand of siRNA in tandem using two independent Pol III promoters (reviewed in Reference (59). After transcription, intrinsic RNase III processes shRNAs into siRNA duplexes followed by incorporation of the siRNA strand into the RNA-induced silencing complex (RISC) (for review see Reference (60). Therefore, both siRNAs and shRNAs are complementary approaches that can silence a targeted gene, and either could be used as method of choice depending on specific situation.