Alternative pre-mRNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few of such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call alternative conserved exons (ACEs), from other orthologous human͞ mouse exons and integrate these features into an exon classification algorithm, ACESCAN. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified Ϸ2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues by using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of ACESCAN predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons that had EST or cDNA evidence of AS in human but were not predicted ACEs, and AS was never observed for 11 negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve the reading frame and less likely to disrupt protein domains than other AS events and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing, and development. Our results also imply that the vast majority of AS events represented in the human EST database are not conserved in mouse.exon skipping ͉ regulatory element ͉ cassette exon ͉ transcriptome ͉ comparative genomics T he processing of human primary transcripts to produce the mRNAs that will direct protein synthesis is often variable, producing multiple alternatively spliced (AS) mRNA products, most commonly by alternative inclusion or exclusion (''skipping'') of individual exons (1-3). Alternative pre-mRNA splicing plays a major role in expanding protein diversity and regulating gene expression in higher eukaryotes (4, 5). Regulated AS is crucial in fruit fly development (3) and in the physiology of the heart, skeletal muscle, brain, and other tissues, and misregulation of AS is associated with human disease (6-8).EST and cDNA sequence databases provide a rich source of information about splicing events occurring in the human and mouse transcriptomes. Considering the set of human ESTs and cDNAs that can be reliably aligned to a human gene locus overlapping a particular exon, this set can be subdivided into transcripts that include the exon and those that exclude, or skip, the exon in question. Here, the skipping of an exon refers to the situation in which a transcript aligns consecutively to an upstream exon and a downstream exon of a gene, omitting the given exon. This consideration can be applied to all of the exons in a human gene, and an analogous subdivision can be made of the mouse transcripts that align to exons of the orthologous mouse gene. Each orthologous human͞mouse exon pair can then be assigned to one of four categories, S H,m , S h,M , S H,M , or S h...

Variation in alternative splicing across human tissues

Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues.

Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality), which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE) activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE)-ESE method. Aligning the single nucleotide polymorphisms (SNPs) from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 ± 0.05). This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype.

We study the nucleotide-nucleotide mutual information function I(k) of the DNA sequences of the three completely sequenced human chromosomes 20, 21, and 22. We find in each human chromosome (i) the absence of the k=3 base pair (bp) sequence periodicity characteristic for protein coding regions, (ii) the absence of the k=10-11 bp sequence periodicity characteristic for both protein secondary structure and DNA bendability, and (iii) the presence of significant statistical dependencies at about k=135 bp and at about k=165 bp. We investigate to which degree the density and composition of interspersed repeats might explain these observed statistical patterns in all three human chromosomes. We use simple stochastic models to substitute known interspersed repeats and find by numerical studies that (iv) the presence of interspersed repeats dominates short-range correlations as measured by I(k) on the scale of several hundred base pairs in human chromosomes 20, 21, and 22. On the other hand, we find that (v) interspersed repeats contribute only weakly to long-range correlations due to the clustering of highly abundant Alu repeats.

RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at .