Alternative Splicing and Disease

Introduction

The hypotheses of Beadle and Tatum (1941), that one gene codes for a single uniquely defined protein, has been disproved many times during the past 30 years by discovering a number of ways how one single gene gives rise to different gene products. The differences may result at the transcriptional level, during RNA pre-processing, m-RNA translation or post-translational protein processing and folding. A recent review by Boeckmann et al. (2005) describes the most important ways of gene product modification.

Alternative splicing increases the diversity of gene products by deriving different final mRNAs from the same pre-mRNA transcript by alternative definition of introns which are spliced out to derive the final mRNA used for translation. But whereas the process of splicing, the interaction of different proteins forming the spliceosome and removing an intron, is investigated and described in great detail by Staley and Guthrie (1998), Will and Luhrmann (2001), Nilsen (2003) and Tazi et al. (2005), the process of how the splice sites are selected is not yet fully understood.

For each single splicing reaction the spliceosome is newly formed in interaction with the pre-mRNA and acts in two basic steps as seen in Figure 1. Cleavage of the donor site and ligation of the 5’ end of the intron to the branch side (step 1) is followed by removal of the intron by cleavage of the acceptor site, the 3’ end of the intron, and the ligation of the neighboring exons (step 2). About hundred interacting molecular splicing factors, many of them are snRNPs, are known to steer this complex process. Four important signals on the pre-mRNA intron are essential to attract these splicing factors to form the spliceosome and to perform splicing. Theses are the donor site at the 5’ end of the intron, the branch point with the nucleotide A located about 17-40 nucleotides upstream of the acceptor site, the polypyrimidine region, and the acceptor site itself which defines the 3’ end of the intron. Exonic and intronic splicing enhancer or silencer sequence motifs additionally affect attraction of all splicing factors. If hit by mutations, all changed sequence signals are prone to change the pattern of splicing and to seriously affect protein expression, eventually causing death and disease.

Figure 1.

The two major steps of splicing; cleavage at the donor site (5’ splice site) and forming the lariat structure (step 1) followed by removing the intron and ligating the exons (step 2)

Krawczak et al. (1992) reported about 15% of all known diseases causing point mutations to directly hit splice sites. This early publication did not consider mutations of exonic or intronic splicing enhancer or silencer signals known to be very important today. The Human Gene Mutation Database published by Stenson et al. 2003 contains 61106 registered mutations of which 5822 affect splicing events. From this set 3633 are point mutations of the GT donor and the AG acceptor splice sites. Skipped exons induced by these mutations often change the reading frame and create aberrant not functional proteins. Mutations within exons, which do not change the resulting proteins, are considered neutral or silent. Still they can damage splicing enhancer or silencer motifs. Studies of the NF1 (Neurofibromatosis type 1) and ATM (Ataxia telangiectasia) mutated gene by Teraoka et al. (1999) and Ars et al. (2000) found that even 50% of the disease causing mutations imply splicing mistakes. Characterization of these splicing defects can help to understand diseases and to find novel strategies for diagnostics and therapy.

Even the normal process of alternative splicing is very complex. Reports by Mironov et al. (1999), Johnson et al. (2003) and Gupta et al. (2004) reported alternative RNA splicing for 35% to 75% of the human genes. Although these numbers must be handled with care, they surely imply that mutations can easily harm the natural balance of alternative splice products and lead to diseases and cancer.