Background Alternative splicing is a mechanism for increasing protein diversity by

Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several SB225002 IC50 of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. Conclusion Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that this more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility. Background Alternative splicing of mRNA transcripts is usually one mechanism by which genomic complexity is usually generated from the surprisingly low number of genes currently estimated from the human genome sequence. The fraction of human genes subject to alternative splicing has risen from 5% in early predictions to at least 75% in a recent genome-wide exon study (Clark et al., in prep.). There are examples of hundreds of alternative splicing events from a single gene, which may affect function by adding or deleting functional domains, changing affinities, and altering mRNA stability. Variable transcripts from a single gene are produced combinatorially through the selection of cassette exons, mutually exclusive exons, retained introns, alternative 3′ or 5′ splice sites, and alternative promoters or polyA sites [1]. Specific alterations in splicing patterns have been found in association with cancers, many of which may play a functional role in transformation, motility and metastasis of tumor tissue. Alternative splicing appears to affect key aspects of neoplasms by altering hormonal signaling, apoptosis and mediators of cell-cell and cell-matrix interactions. Modifications in functionality may be generated, for example, through the deletion of a signaling domain, increased affinity for messenger ligands, or change in affinity or activity toward extracellular components. The latter frequently results in increased cell migration and invasion (For general reviews, see [2,3]). Although in most cases the relationship between specific splicing events and the etiology of cancer is largely unproven, alternative splicing presents novel targets for SB225002 IC50 diagnostic and therapeutic measures. Abnormal splicing of several genes has been observed in tumors of colorectal origin, including CD44 [4,5], MUC2 [6], SRF [7], NCAM [8], MLH, MSH [9] and members of the Wnt pathway [10]. Some Rabbit Polyclonal to THBD of these alternately spliced gene products may have therapeutic utility as markers of the progression of the disease or as drug targets; however there are insufficient data supporting their relevance in larger clinical populations. Individual patient differences, tissue complexity and lack of tools for comprehensive analysis of splice variation have made the task challenging. We have analyzed both differential gene expression and alternative splicing in a small collection of colon cancers employing a microarray (GeneChip Human Exon 1.0 ST) targeting over a million putative exons. The exons can be virtually reassembled into over 250,000 transcripts according to a range of annotation sources. The inclusiveness of the array design allowed a flexible gene expression analysis, initially targeting high confidence transcripts and then extending to a more speculative set. Cancer-specific splice variations were detected with experimental algorithms and candidate events were subsequently validated with RT-PCR. We found several splice variants, some of which have been previously associated with cancer, and several which have SB225002 IC50 not. The majority of the identified splicing events affect mediators of cytoskeletal organization, the extracellular matrix or integrin signaling, and may be involved in cell migration and invasion. Results Array Design The SB225002 IC50 GeneChip Human Exon 1.0 ST array was designed to be as inclusive as possible at the exon level, deriving from annotations ranging from empirically decided, highly curated mRNA sequences to ab-initio computational predictions [11]. The array contains approximately 5.4 million 5-m features (probes) grouped into 1.4 million probesets interrogating over one million exon clusters (exon annotations from various sources that overlap by genomic.

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