Cellular functions depend on numerous protein-coding and non-coding RNAs and the RNA-binding proteins associated with them, which form ribonucleoprotein complexes (RNPs). Mutations that disrupt either the RNA or protein components of RNPs or the factors required for their assembly can be deleterious. Alternative splicing provides cells with an exquisite capacity to fine-tune their transcriptome and proteome in response to cues. Splicing depends on a complex code, numerous RNA-binding proteins and an enormously intricate network of interactions among them, increasing the opportunity for exposure to mutations and mis-regulation that cause disease. The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry is providing new RNA-based tools for developing therapeutics.

The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977;Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999;Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002). Constitutive splicing and the basal splicing machineryThe typical human gene contains an average of 8 exons. Internal exons average 145 nucleotides (nt) in length, and introns average more than 10 times this size and can be much larger (Lander et al. 2001). Exons are defined by rather short and degenerate classical splice-site sequences at the intron/exon borders (5Ј splice site, 3Ј splice site, and branch site; Fig. 1A). Components of the basal splicing machinery bind to the classical splice-site sequences and promote assembly of the multicomponent splicing complex known as the spliceosome. The spliceosome performs the two primary functions of splicing: recognition of the intron/exon boundaries and catalysis of the cut-and-paste reactions that remove introns and join exons. The spliceosome is made up of five small nuclear ribonucleoproteins (snRNPs) and >100 proteins. Each snRNP is composed of a single uridinerich small nuclear RNA (snRNA) and multiple proteins. The U1 snRNP binds the 5Ј splice site, and the U2 snRNP binds the branch site via RNA:RNA interactions between the snRNA and the pre-mRNA (Fig. 1B). Spliceosome assembly is highly dynamic in that complex rearrangements of RNA:RNA, RNA:protein, and protein:protein interactions take place within the spliceosome. Coinciding with these internal rearrangements, both splice sites are recognized multiple times by interactions with different components during the course of spliceosome assembly (for example, see Burge et al. 1999;Du and Rosbash 2002;Lallena et al. 2002;Liu 2002). The catalytic component is likely to be U6 snRNP, which joins the spliceosome as a U4/U6 · U5 tri-snRNP (Villa et al. 2002).A splicing error that adds or removes even 1 nt will disrupt the open reading frame of an mRNA; yet exons are correctly spliced from within tens of thousands of intronic nucleotides. This remarkable precision is, in part, built into the mechanism of intron removal because once...

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.

Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases, and recent studies have provided new insights into the mechanisms by which these effects occur. An unexpectedly large fraction of exonic mutations exhibit a primary pathogenic effect on splicing. Furthermore, normal genetic variation significantly contributes to disease severity and susceptibility by affecting splicing efficiency.