Computational analysis of alternative splicing using EST tissue information

Expressed sequence tags (ESTs) from normal and tumor tissues have been deposited in public databases. These ESTs and all mRNA sequences were aligned with the human genome sequence using LEADS, Compugen's alternative splicing modeling platform. We developed a novel computational approach to analyze tissue information of aligned ESTs in order to identify cancer-specific alternative splicing and gene segments highly expressed in particular cancers. Several genes, including one encoding a possible pre-mRNA splicing factor, displayed cancer-specific alternative splicing. In addition, multiple candidate gene segments highly expressed in colon cancers were identified.     

Genome-wide detection of tissue-specific alternative splicing in the human transcriptome

We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye-retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10-30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.     

Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes

Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue-specific isoforms of a target gene that expedites a highly conserved signaling pathway.     

Splicing factor and exon profiling across human tissues

It has been shown that alternative splicing is especially prevalent in brain and testis when compared to other tissues. To test whether there is a specific propensity of these tissues to generate splicing variants, we used a single source of high-density microarray data to perform both splicing factor and exon expression profiling across 11 normal human tissues. Paired comparisons between tissues and an original exon-based statistical group analysis demonstrated after extensive RT-PCR validation that the cerebellum, testis, and spleen had the largest proportion of differentially expressed alternative exons. Variations at the exon level correlated with a larger number of splicing factors being expressed at a high level in the cerebellum, testis and spleen than in other tissues. However, this splicing factor expression profile was similar to a more global gene expression pattern as a larger number of genes had a high expression level in the cerebellum, testis and spleen. In addition to providing a unique resource on expression profiling of alternative splicing variants and splicing factors across human tissues, this study demonstrates that the higher prevalence of alternative splicing in a subset of tissues originates from the larger number of genes, including splicing factors, being expressed than in other tissues.     

Neural Differentiation Modulates the Vertebrate Brain Specific Splicing Program

Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation.     

CNS-Specific Ribozyme Expression

Targeted genetic deletion is a powerful tool for analysis of gene function, but the standard approaches carry certain inescapable disadvantages. First, deletion is ubiquitous; tissue-specific knockout cannot be obtained. Second, temporal regulation of depletion is unattainable; the deleted functions are absent throughout the animal's development. As a consequence, during ontogeny, other gene products may be able to compensate, filling the functional gap. Furthermore bifunctional proteins exist that fulfill one role during development and another in the mature organism; deletion will remove the early function and, if this is lethal, the later function will remain undetected. Third, if genes utilize alternative splicing to control protein expression, it is difficult to target one spliced mRNA while leaving intact its related, but different, siblings. We review how these problems may be circumvented using ribozymes to diminish gene expression in a tissue-specific and temporally regulated manner and provide guidelines for the design and delivery of active ribozymesin vivo.Such methods may be particularly useful for analysis of genes involved in ontogeny and function of the central nervous system, in which individual genes may be expressed with alternative splicing patterns, or at differentially regulated levels, at different stages of CNS development.     

Overexpression of a family of RPEL proteins modifies cell shape

Proteins containing RPEL motifs (e.g., MAL) are important in the regulation of gene expression by the actin cytoskeleton. Screening the ENSEMBL database for RPEL proteins identified four additional proteins that contain RPEL motifs and nuclear localisation sequences, three of which (RPEL-A, RPEL-B and RPEL-C) are expressed in adult mouse tissues with different expression profiles. The mRNAs encoding RPEL-B and RPEL-C were subject to alternative splicing. Expression of these genes in cells indicated that they had a marked effect on cell shape. Furthermore, when expressed with a nuclear localised actin all of the different forms became restricted to the nucleus.     

Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing

All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA (pre-mRNA) splicing. Recent computational analyses predict that most human exons contain elements required for splicing coinciding with an appreciation for the high frequency with which mutations that disruption pre-mRNA splicing cause disease. Minigenes provide a means to directly determine whether disease-causing mutations or single nucleotide polymorphisms (SNPs) affect splicing efficiency. Minigenes have also been instrumental in investigations of alternative splicing to identify cis elements required for cell-specific splicing events, demonstrating regulation of individual splicing events by specific RNA binding proteins, and correlating binding of these splicing regulators with splicing regulation. Here we present a versatile minigene plasmid vector designed for rapid cloning and analysis of cis elements and trans-acting factors that influence splicing efficiency or regulate cell-specific splicing. Ubiquitous expression and unique restriction sites allow for straightforward replacement of a variety of gene segments to analyze the effects of nucleotide substitutions on splicing, to identify tissue-specific regulatory elements, or to determine responsiveness to coexpressed proteins or small molecules.