Pre-mRNA splicing is a sophisticated and ubiquitous nuclear process, which is a natural source of cancer-causing errors in gene expression. Intronic splice site mutations of tumor suppressor genes often cause exon-skipping events that truncate proteins just like classical nonsense mutations. Also, many studies over the last 20 years have reported cancerspecific alternative splicing in the absence of genomic mutations. Affected proteins include transcription factors, cell signal transducers, and components of the extracellular matrix. Antibodies against alternatively spliced products on cancer cells are currently in clinical trials, and competitive reverse transcription-PCR across regions of alternative splicing is being used as a simple diagnostic test. As well as being associated with cancer, the nature of the alternative gene products is usually consistent with an active role in cancer; therefore, the alternative splicing process itself is a potential target for gene therapy. Splice Site Mutations Can Cause Aberrant Splicing and CancerDefects in mRNA splicing are an important cause of disease (1-3). The most common form of splicing defects are genomic splice site point mutations, and a recent survey found 29 different p53 splice site mutations in Ͼ12 different types of cancer (4). Ninety-nine percent of all exons are flanked by the intronic dinucleotides GT and AG at the 5Ј and 3Ј splice sites respectively, and mutation of these sites usually causes exclusion of the adjacent exon (Fig. 1A), although sometimes splice site mutations have even more drastic consequences, e.g., double exon skipping in MLH1 in hereditary nonpolyposis colorectal cancer (5). More than half of all exon deletions lead to truncation of the encoded protein and as such, mutations in tumor suppressor genes at the invariant intronic dinucleotide can act much as classical nonsense mutations. For example a GT to AT 5Ј splice site mutation in hSNF5 causes deletion of exon 7, a frame shift, and a truncated reading frame, and this causes infant brain tumors when a "second hit" is provided at the wild-type allele by a deletion (6). Similarly a 3Ј splice site AG to AT mutation caused constitutive loss of exon 4 of the APC gene in colorectal to liver metastases, where the wild-type allele was deleted (ref. 7; Fig. 1A).Mutations in the less conserved splice site consensus away from the invariant dinucleotides tend to lead to partial aberrant splicing, often with a relatively mild phenotype. For example, the most common pathogenic mutation of the ATM gene is linked to breast cancer but is incompletely penetrant and is thought to have originated in Palaeolithic times. This is a mutation at the sixth position of an intron that causes a proportion of transcripts to skip an exon and to be frame shifted leading to a truncated protein (8). The polypyrimidine tract signal associated with the 3Ј splice site is another hot spot for mutation (Fig. 1A). An intronic mutation 11 nucleotides upstream of a 3Ј splice site in MLH1 knocks out an exon, and this ...

Alternative splicing expands the coding capacity of metazoan genes, and it was largely genetic studies in the fruit-fly Drosophila melanogaster that established the principle that regulated alternative splicing results in tissue- and stage-specific protein isoforms with different functions in development. Alternative splicing is particularly prominent in germ cells, muscle and the central nervous system where it modulates the expression of various proteins including cell-surface molecules and transcription factors. Studies in flies have given us numerous insights into alternative splicing in terms of upstream regulation, the exquisite diversity of their forms and the key differential cellular functions of alternatively spliced gene products. The current inundation of transcriptome sequencing data from Drosophila provides an unprecedented opportunity to gain a comprehensive view of alternative splicing.

Alternative splicing of pre-mRNA increases the diversity of protein functions. Here we show that about half of all active alternative splicing events in ovarian and breast tissues are changed in tumors, and many seem to be regulated by a single factor; sequence analysis revealed binding sites for the RNA binding protein FOX2 downstream of one-third of the exons skipped in cancer. High-resolution analysis of FOX2 binding sites defined the precise positions relative to alternative exons at which the protein may function as either a silencer or an enhancer. Most of the identified targets were shifted in the same direction by FOX2 depletion in cell lines as they were in breast and ovarian cancer tissues. Notably, we found expression of FOX2 itself is downregulated in ovarian cancer and its splicing is altered in breast cancer samples. These results suggest that the decreased expression of FOX2 in cancer tissues modulates splicing and controls proliferation.

Alternative pre-mRNA splicing leads to distinct products of gene expression in development and disease. Antagonistic splice variants of genes involved in differentiation, apoptosis, invasion and metastasis often exist in a delicate equilibrium that is found to be perturbed in tumours. In several recent examples, splice variants that are overexpressed in cancer are expressed as hyper-oncogenic proteins, which often correlate with poor prognosis, thus suggesting improved diagnosis and follow up treatment. Global gene expression technologies are just beginning to decipher the interplay between alternatively spliced isoforms and protein-splicing factors that will lead to identification of the mutations in these trans-acting factors responsible for pathogenic alternative splicing in cancer.