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PubMed2005

Bioinformatics analysis of alternative splicing.

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.

Algorithms↗

PubMed1993

General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer.

The general splicing factor SF2/ASF binds in a sequence-specific manner to a purine-rich exonic splicing enhancer (ESE) in the last exon of bovine growth hormone (bGH) pre-mRNA. More importantly, SF2/ASF stimulates in vitro splicing of bGH intron D through specific interaction with the ESE sequences. However, another general splicing factor, SC35, does not bind the ESE sequences and has no effect on bGH intron D splicing. Thus, one possible function of SF2/ASF in alternative and, perhaps, constitutive pre-mRNA splicing is to recognize ESE sequences. The stimulation of bGH intron D splicing by SF2/ASF is counteracted by the addition of hnRNP A1. The relative levels of SF2/ASF and hnRNP A1 influence the efficiency of bGH intron D splicing in vitro and may be the underlying mechanism of this alternative pre-mRNA processing event in vivo.

Alternative Splicing↗

PubMed2001

Alternative splicing and genome complexity.

Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. It has recently been proposed as a mechanism by which higher-order diversity is generated. Here we show, using large-scale expressed sequence tag (EST) analysis, that among seven different eukaryotes the amount of alternative splicing is comparable, with no large differences between humans and other animals.

Alternative Splicing↗

PubMed2003

ASAP: the Alternative Splicing Annotation Project.

Recently, genomics analyses have demonstrated that alternative splicing is widespread in mammalian genomes (30-60% of genes reported to have multiple isoforms), and may be one of their most important mechanisms of functional regulation. However, by comparison with other genomics data such as genome annotation, SNPs, or gene expression, there exists relatively little database infrastructure for the study of alternative splicing. We have constructed an online database ASAP (the Alternative Splicing Annotation Project) for biologists to access and mine the enormous wealth of alternative splicing information coming from genomics and proteomics. ASAP is based on genome-wide analyses of alternative splicing in human (30 793 alternative splice relationships found) from detailed alignment of expressed sequences onto the genomic sequence. ASAP provides precise gene exon-intron structure, alternative splicing, tissue specificity of alternative splice forms, and protein isoform sequences resulting from alternative splicing. Moreover, it can help biologists design probe sequences for distinguishing specific mRNA isoforms. ASAP is intended to be a community resource for collaborative annotation of alternative splice forms, their regulation, and biological functions. The URL for ASAP is http://www.bioinformatics.ucla.edu/ASAP.

Alternative Splicing↗

PubMed2005

Evolutionarily conserved and diverged alternative splicing events show different expression and functional profiles.

To better decipher the functional impact of alternative splicing, we classified alternative splicing events in 10,818 pairs of human and mouse genes based on conservation at genome and transcript levels. Expression levels of conserved alternative splices in human and mouse expressed sequence tag databases show strong correlation, indicating that alternative splicing is similarly regulated in both species. A total of 43% (8921) of mouse alternative splices could be found in the human genome but not in human transcripts. Five of eleven tested mouse predictions were observed in human tissues, demonstrating that mouse transcripts provide a valuable resource for identifying alternative splicing events in human genes. Combining gene-specific measures of conserved and diverged alternative splicing with both gene classification based on Gene Ontology (GO) and microarray-determined gene expression in 52 diverse human tissues and cell lines, we found conserved alternative splicing most enriched in brain-expressed signaling pathways. Diverged alternative splicing is more prevalent in testis and cancerous cell line up-regulated processes, including protein biosynthesis, responses to stress and responses to endogenous stimuli. Using conservation as a surrogate for functional significance, these results suggest that alternative splicing plays an important role in enhancing the functional capacity of central nervous systems, while non-functional splicing more frequently occurs in testis and cell lines, possibly as a result of cellular stress and rapid proliferation.

Alternative Splicing↗

PubMed2000

Role of SR protein modular domains in alternative splicing specificity in vivo.

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.

Adenovirus E1A Proteins↗

PubMed1998

Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA.

Splicing of pre-mRNA transcripts is regulated by consensus sequences at intron (intervening sequence, IVS) boundaries and the branch site. In vitro studies have shown that the small introns of some genes also require intron splice enhancers (ISE) to modulate splice site selection. An autosomal dominant form of isolated GH deficiency (IGHD-II) is caused by mutations in IVS3 of the GH-1 gene that cause exon 3 (E3) skipping, resulting in truncated hGH products that prevent secretion of normal hGH. Interestingly, some of these IGHD-II mutations perturb an ISE that is buried in IVS3. We localized this ISE by quantitating the effects of deletions within IVS3 on E3 skipping. The importance of individual nucleotides to ISE function was determined by analyzing the effects of point mutants and additional deletions. Our results show that (i) an ISE with a G2X1-4G3motif resides in IVS3 of GH-1; (ii) both runs of Gs are required for ISE function; (iii) a single copy of the ISE regulates E3 skipping and (iv) ISE function can be modified by an adjacent AC element. Our findings reveal a new mechanism by which mutations can cause inherited human endocrine disorders and suggest that (i) ISEs may regulate splicing of transcripts of other genes and (ii) mutations of these ISEs or of the trans -acting factors that bind them may cause other genetic disorders.

Alternative Splicing↗

PubMed2001

Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing.

Nascent transcripts are the true substrates for many splicing events in mammalian cells. In this review we discuss transcription, splicing, and alternative splicing in the context of co-transcriptional processing of pre-mRNA. The realization that splicing occurs co-transcriptionally requires two important considerations: First, the cis-acting elements in the splicing substrate are synthesized at different times in a 5' to 3' direction. This dynamic view of the substrate implies that in a 100 kb intron the 5' splice site will be synthesized as much as an hour before the 3' splice site. Second, the transcription machinery and the splicing machinery, which are both complex and very large, are working in close proximity to each other. It is therefore likely that these two macromolecular machines interact, and recent data supporting this notion is discussed. We propose a model for co-transcriptional pre-mRNA processing that incorporates the concepts of splice site-tethering and dynamic exon definition. Also, we present a dynamic view of the alternative splicing of FGF-R2 and suggest that this view could be generally applicable to many regulated splicing events.

Alternative Splicing↗

PubMed2002

The human XPC DNA repair gene: arrangement, splice site information content and influence of a single nucleotide polymorphism in a splice acceptor site on alternative splicing and function.

XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82-882 bp) and 15 introns (0.08-5.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcription-polymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in approximately 0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in approximately 0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (-0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (approximately 2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility.

Alternative Splicing↗

PubMed2004

[Molecular mechanism of mRNA alternative splicing].

Eukayotic pre-mRNAs are spliced to form mature mRNA. The pre-mRNAs alternative splicing greatly increases the diversity of proteins and the complexity of genes expression. The recognition of splice sites can occur across intron (intron definition) or across exon (exon definition). There are many kinds of alternative splicing patterns, including the choice of alternative splicing sites, the choice of alternative splicing ends, the alternative splicing of mutually exclusive exons, and an intron can be excised from or retained in mRNA. The splice sites choice process is regulated by many kinds of cis-acting and trans-acting elements and is closely related with the basic splicing process. Some of the splicing factors in basic splicing process can regulate the alternative splicing. The alternative splicing is also a co-transcriptional process. Promoters can regulate alternative splicing and produce different mRNA isoforms. Many molecular mechanisms of alternative splicing have been proposed, and it was also found that the RNA editing and trans-splicing could also regulate alternative splicing.

Alternative Splicing↗

PubMed2004

CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific splicing enhancer-dependent alternative splicing.

We previously described a family of five RNA-binding proteins: CUG-binding protein, embryonic lethal abnormal vision-type RNA-binding protein 3, and the CUG-binding protein and embryonic lethal abnormal vision-type RNA-binding protein 3-like factors (CELFs) 3, 4, and 5. We demonstrated that all five of these proteins specifically activate exon inclusion of cardiac troponin T minigenes in vivo via muscle-specific splicing enhancer (MSE) sequences. We also predicted that a sixth family member, CELF6, was located on chromosome 15. Here, we describe the isolation and characterization of CELF6. Like the previously described CELF proteins, CELF6 shares a domain structure containing three RNA-binding domains and a divergent domain of unknown function. CELF6 is strongly expressed in kidney, brain, and testis and is expressed at very low levels in most other tissues. In the brain, expression is widespread and maintained from the fetus to the adult. CELF6 activates exon inclusion of a cardiac troponin T minigene in transient transfection assays in an MSE-dependent manner and can activate inclusion via multiple copies of a single element, MSE2. These results place CELF6 in a functional subfamily of CELF proteins that includes CELFs 3, 4, and 5. CELF6 also promotes skipping of exon 11 of insulin receptor, a known target of CELF activity that is expressed in kidney.

Alternative Splicing↗

PubMed2004

Detecting tissue-specific regulation of alternative splicing as a qualitative change in microarray data.

Alternative splicing has recently emerged as a major mechanism of regulation in the human genome, occurring in perhaps 40-60% of human genes. Thus, microarray studies of functional regulation could, in principle, be extended to detect not only the changes in the overall expression of a gene, but also changes in its splicing pattern between different tissues. However, since changes in the total expression of a gene and changes in its alternative splicing can be mixed in complex ways among a set of samples, separating these effects can be difficult, and is essential for their accurate assessment. We present a simple and general approach for distinguishing changes in alternative splicing from changes in expression, based on detecting systematic anti-correlation between the log-ratios of two different samples versus a pool containing both samples. We have tested this analysis method on microarray data for five human tissues, generated using a standard microarray platform and experimental protocols shown previously to be sensitive to alternative splicing. Our automatic analysis was able to detect a wide variety of tissue-specific alternative splicing events, such as exon skipping,mutually exclusive exons, alternative 3' and alternative 5' splicing, alternative initiation and alternative termination, all of which were validated by independent reverse-transcriptase PCR experiments, with validation rates of 70-85%. Our analysis method also enables hierarchical clustering of genes and samples by the level of similarity to their alternative splicing patterns, revealing patterns of tissue-specific regulation that are distinct from those obtained by hierarchical clustering of gene expression from the same microarray data. Our data and analysis source code are available from http://www.bioinformatics.ucla.edu/ASAP.

Alternative Splicing↗

PubMed2004

Identification of alternative splicing regulators by RNA interference in Drosophila.

Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing.

Alternative Splicing↗

PubMed2004

The regulation and regulatory activities of alternative splicing of the SMN gene.

Alternative splicing is an essential process that produces protein diversity in humans. It is also the cause of many complex diseases. Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder, is caused by the absence of or mutations in the Survival Motor Neuron 1 (SMN1) gene, which encodes an essential protein. A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 because exon 7 is alternatively spliced, producing a truncated protein, which is unstable. SMN1 and SMN2 differ by a critical C-to-T substitution at position 6 of exon 7 in SMN2 (C6U transition in mRNA). This substitution alone is enough to cause an exon 7 exclusion in SMN2. Various cis- and trans-acting factors have been shown to neutralize the inhibitory effects of C6U transition. Published reports propose models in which either abrogation of an enhancer element associated with SF2/ASF or gain of a silencer element associated with hnRNP A1 is the major cause of exon 7 exclusion in SMN2. Most recent model suggests the presence of an EXtended INhibitory ContexT (Exinct) that is formed as a consequence of C6U transition in exon 7 of SMN2. In Exinct model, several factors may affect exon 7 splicing through cooperative interactions. Such regulation may be common to many alternatively spliced exons in humans. Recent advances in our understanding of SMN gene splicing reveals multiple challenges that are specific to in vivo regulation, which we now know is intimately connected with other biological pathways.

Alternative Splicing↗

PubMed1997

Alternative splicing of Pax6 in bovine eye and evolutionary conservation of intron sequences.

Pax-6 is essential for eye development and for tissue-specific gene expression within the eye. Splicing of an alternative exon (5a) leads to variant DNA binding specificity in Pax6. Here we show that in the mature bovine eye the relative abundance of the alternative Pax6-5a mRNA varies markedly, particularly between the lens and iris. Additional alternative splicing products were identified which may lead to "domain swapping" or truncation of Pax6 proteins. Comparison of amphibian (Xenopus) and mammalian (bovine) Pax-6 genes revealed highly conserved intronic sequences flanking exon 5a, including potential lariat sites which may have a role in the mechanism of alternative splicing.

Alternative Splicing↗

PubMed2006

The ASAP II database: analysis and comparative genomics of alternative splicing in 15 animal species.

We have greatly expanded the Alternative Splicing Annotation Project (ASAP) database: (i) its human alternative splicing data are expanded approximately 3-fold over the previous ASAP database, to nearly 90,000 distinct alternative splicing events; (ii) it now provides genome-wide alternative splicing analyses for 15 vertebrate, insect and other animal species; (iii) it provides comprehensive comparative genomics information for comparing alternative splicing and splice site conservation across 17 aligned genomes, based on UCSC multigenome alignments; (iv) it provides an approximately 2- to 3-fold expansion in detection of tissue-specific alternative splicing events, and of cancer versus normal specific alternative splicing events. We have also constructed a novel database linking orthologous exons and orthologous introns between genomes, based on multigenome alignment of 17 animal species. It can be a valuable resource for studies of gene structure evolution. ASAP II provides a new web interface enabling more detailed exploration of the data, and integrating comparative genomics information with alternative splicing data. We provide a set of tools for advanced data-mining of ASAP II with Pygr (the Python Graph Database Framework for Bioinformatics) including powerful features such as graph query, multigenome alignment query, etc. ASAP II is available at http://www.bioinformatics.ucla.edu/ASAP2.

Alternative Splicing↗

PubMed2006

Intronic alternative splicing regulators identified by comparative genomics in nematodes.

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.

Alternative Splicing↗

PubMed2002

Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca(v)alpha1.

Alternative splicing is a critical mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes. This review focuses on recent studies of voltage-gated calcium (Ca) channel Ca(v)alpha1 subunit splice isoforms in neurons. Voltage-gated Ca channels couple changes in neuronal activity to rapid changes in intracellular Ca levels that in turn regulate an astounding range of cellular processes. Only ten genes have been identified that encode Ca(v)alpha1 subunits, an insufficient number to account for the level of functional diversity among voltage-gated Ca channels. The consequences of regulated alternative splicing among the genes that comprise voltage-gated Ca channels permits specialization of channel function, optimizing Ca signaling in different regions of the brain and in different cellular compartments. Although the full extent of alternative splicing is not yet known for any of the major subtypes of voltage-gated Ca channels, it is already clear that it adds a rich layer of structural and functional diversity".

Alternative Splicing↗