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Results for “Alternative Splicing”

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PubMed2002

Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels.

Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels.

Alternative Splicing↗

PubMed2005

Creation and disruption of protein features by alternative splicing -- a novel mechanism to modulate function.

BACKGROUND: Alternative splicing often occurs in the coding sequence and alters protein structure and function. It is mainly carried out in two ways: by skipping exons that encode a certain protein feature and by introducing a frameshift that changes the downstream protein sequence. These mechanisms are widespread and well investigated. RESULTS: Here, we propose an additional mechanism of alternative splicing to modulate protein function. This mechanism creates a protein feature by putting together two non-consecutive exons or destroys a feature by inserting an exon in its body. In contrast to other mechanisms, the individual parts of the feature are present in both splice variants but the feature is only functional in the splice form where both parts are merged. We provide evidence for this mechanism by performing a genome-wide search with four protein features: transmembrane helices, phosphorylation and glycosylation sites, and Pfam domains. CONCLUSION: We describe a novel type of event that creates or removes a protein feature by alternative splicing. Current data suggest that these events are rare. Besides the four features investigated here, this mechanism is conceivable for many other protein features, especially for small linear protein motifs. It is important for the characterization of functional differences of two splice forms and should be considered in genome-wide annotation efforts. Furthermore, it offers a novel strategy for ab initio prediction of alternative splice events.

Alternative Splicing↗

PubMed2000

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1.

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.

Alternative Splicing↗

PubMed2002

Finding signals that regulate alternative splicing in the post-genomic era.

Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification.

Alternative Splicing↗

PubMed1998

Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation.

Alternative splicing of pre-mRNAs is a powerful and versatile regulatory mechanism that can effect quantitative control of gene expression and functional diversification of proteins. It contributes to major developmental decisions and also to fine tuning of gene function. Genetic and biochemical approaches have identified cis-acting regulatory elements and trans-acting factors that control alternative splicing of specific pre-mRNAs. Both approaches are contributing to an understanding of their mode of action. Some alternative splicing decisions are controlled by specific factors whose expression is highly restricted during development, but others may be controlled by more modest variations in the levels of general factors acting cooperatively or antagonistically. Certain factors play active roles in both constitutive splicing and regulation of alternative splicing. Cooperative and antagonistic effects integrated at regulatory elements are likely to be important for specificity and for finely tuned differences in cell-type-specific alternative splicing patterns.

Alternative Splicing↗

PubMed2004

Alternate splice variants of mouse NR2B gene.

Two alternate spliced transcripts of N-methyl-D-aspartate receptor (NR2B) gene derived from a common promoter have been reported earlier. In the present study, we have examined the expression of alternative splice variants of NR2B gene using 5' rapid amplification of C terminal end (RACE) in the RNA derived from mouse cortex. RT-PCR was performed to confirm the RACE data. Here we report the occurrence of three transcripts containing a novel alternate spliced exon, we refer to as exon 1'. This new exon is located upstream of the transcription start site identified by Klein et al. [Gene 208 (1998) 259] in the 5' untranslated region (UTR) of NR2B, thus making a new alternate 5' end of the NR2B transcript. The molecular nature of the transcripts containing the new exon appears to be highly heterogeneous. These results are interesting in that they are of potential importance in translational regulation of the NR2B gene.

5' Untranslated Regions↗

PubMed2006

Variable window binding for mutually exclusive alternative splicing.

BACKGROUND: Genes of advanced organisms undergo alternative splicing, which can be mutually exclusive, in the sense that only one exon is included in the mature mRNA out of a cluster of alternative choices, often arranged in a tandem array. In many cases, however, the details of the underlying biologic mechanisms are unknown. RESULTS: We describe 'variable window binding'--a mechanism used for mutually exclusive alternative splicing by which a segment ('window') of a conserved nucleotide 'anchor' sequence upstream of the exon 6 cluster in the pre-mRNA of the fruitfly Dscam gene binds to one of the introns, thereby activating selection of the exon directly downstream from the binding site. This mechanism is supported by the fact that the anchor sequence can be inferred solely from a comparison of the intron sequences using a genetic algorithm. Because the window location varies for each exon choice, regulation can be achieved by obstructing part of that sequence. We also describe a related mechanism based on competing pre-mRNA stem-loop structures that could explain the mutually exclusive choice of exon 17 of the Dscam gene. CONCLUSION: On the basis of comparative sequence analysis, we propose efficient biologic mechanisms of alternative splicing of the Drosophila Dscam gene that rely on the inherent structure of the pre-mRNA. Related mechanisms employing 'locus control regions' could be involved on other occasions of mutually exclusive choices of exons or genes.

Algorithms↗

PubMed2004

Identification of alternatively spliced mRNA variants related to cancers by genome-wide ESTs alignment.

Several databases have been published to predict alternative splicing of mRNAs by analysing the exon linkage relationship by alignment of expressed sequence tags (ESTs) to the genome sequence; however, little effort has been made to investigate the relationship between cancers and alternative splicing. We developed a program, Alternative Splicing Assembler (ASA), to look for splicing variants of human gene transcripts by genome-wide ESTs alignment. Using ASA, we constructed the biosino alternative splicing database (BASD), which predicted splicing variants for reference sequences from the reference sequence database (RefSeq) and presented them in both graph and text formats. EST clusters that differ from the reference sequences in at least one splicing site were counted as splicing variants. Of 4322 genes screened, 3490 (81%) were observed with at least one alternative splicing variants. To discover the variants associated with cancers, tissue sources of EST sequences were extracted from the UniLib database and ESTs from the same tissue type were counted. These were regarded as the indicators for gene expression level. Using Fisher's exact test, alternative splicing variants, of which EST counts were significantly different between cancer tissues and their counterpart normal tissues, were identified. It was predicted that 2149 variants, or 383 variants after Bonferroni correction, of 26 812 variants were likely tumor-associated. By reverse transcription-PCR, 11 of 13 novel alternative splicing variants and eight of nine variants' tissue specificity were confirmed in hepatocellular carcinoma and in lung cancer. The possible involvement of alternative splicing in cancer is discussed.

Alternative Splicing↗

PubMed2005

Alternative splicing of Pot1 (Protection of telomere)-like genes in Arabidopsis thaliana.

The Pot1 (Protection of telomere 1) is a G-rich single-stranded telomeric DNA binding protein, identified first in Schizosaccharomyces pombe, and shown to play an important role in stabilizing chromosomes. Pot1-like proteins or their encoding genes have been identified from yeasts to mammals. Based on the N-terminal amino acid sequences of fission yeast and human Pot1, two Pot1-like proteins (AtPOT1-1 and AtPOT1-2) have been identified in Arabidopsis thaliana, but neither of them has been characterized yet. In this study, we amplified their full-length cDNAs by RT-PCR and found three different variants for AtPOT1-1 and two for AtPOT1-2 genes, suggesting that they are exposed to alternative splicing. Alternative splicing also occurs in human Pot1, and only one out of five splicing variants had tissue specificity. However, no tissue specificity was found for any variants of the AtPOT1-1 and AtPOT1-2 genes among buds, flowers, leaves, roots, stems, siliques and cultured cells. Northern blot hybridization indicated that AtPOT1-1 expresses more in meristematic tissues than in vegetative tissues. By western blot analysis, we found that the antibody made against the N-terminal amino acids of AtPOT1-1 recognized three different polypeptides, indicating that all three variants are being translated in Arabidopsis.

Alternative Splicing↗

PubMed1997

Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

Alternative Splicing↗

PubMed2025

Alternative Splicing in Mechanically Stretched Podocytes as a Model of Glomerular Hypertension.

KEY POINTS: Mechanical stretch induced over 3000 alternative splicing events in podocytes, affecting gene expression and protein abundance. Seventeen genes showed consistent splicing events across multiple analysis tools, with key isoform changes. Shroom3 and Myl6 underwent isoform switches under mechanical stretch, altering the C-terminal sequence and interaction properties of Myl6. BACKGROUND: Alterations in pre-mRNA splicing are crucial to the pathophysiology of various diseases. However, the effects of alternative splicing of mRNA on podocytes in hypertensive nephropathy are still unknown. The Sys_CARE project aimed to identify alternative splicing events involved in the development and progression of glomerular hypertension. METHODS: Murine podocytes were exposed to mechanical stretch, after which proteins and mRNA were analyzed by proteomics, RNA sequencing, and several bioinformatic alternative splicing tools. RESULTS: Using transcriptomic and proteomic analysis, we identified significant changes in gene expression and protein abundance because of mechanical stretch. RNA-Seq identified over 3000 alternative spliced genes after mechanical stretch, including all types of alternative splicing events. Among these, 17 genes exhibited an alternative splicing event across four different splicing analysis tools. From this group, we focused on Myl6, a component of the myosin protein complex, and Shroom3, an actin-binding protein essential for podocyte function. We identified two Shroom3 isoforms with significant expression changes under mechanical stretch, which was validated by quantitative RT-PCR and in situ hybridization. In addition, we observed an expression switch of two Myl6 isoforms after mechanical stretch, accompanied by an alteration in the C-terminal amino acid sequence. CONCLUSIONS: A comprehensive RNA-Seq analysis of mechanically stretched podocytes identified novel potential podocyte-specific biomarkers and highlighted significant alternative splicing events, notably in the mRNA of Shroom3 and Myl6.

podocyte↗

PubMed2005

Characterization and prediction of alternative splice sites.

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC content, U2AF35 binding site score, and oligonucleotide frequencies. For the predictive classification of splice sites, pattern recognition models for different splicing factor binding sites and oligonucleotide frequency models (OFMs) were combined using backpropagation networks. 67.45% of acceptor sites and 71.23% of donor sites are correctly classified by networks trained for classification of constitutive and alternative isoform/cryptic splice sites. A web-application for the prediction of alternative splice sites is available at http://es.embnet.org/~mwang/assp.html .

Alternative Splicing↗

PubMed2004

Alternatively spliced human genes by exon skipping--a database (ASHESdb).

UNLABELLED: Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. Exon skipping is the most commonly known alternative splicing mechanism. A comprehensive database of alternative splicing by exon skipping is made available for the human genome data. 1,229 human genes are identified to exhibit alternative splicing by exon skipping. AVAILABILITY: http://sege.ntu.edu.sg/wester/ashes/.

Alternative Splicing↗

PubMed1995

Gene therapeutic approach to primary and metastatic brain tumors: I. CD44 variant pre-RNA alternative splicing as a CEPT control element.

Our laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible for the generation of CD44 variant (CD44v) isoforms. Based on clear differences in the expression of these CD44v isoforms between normal and malignant tissues, we believe that elucidation of the mechanisms underlying the regulation of CD44 alternative splicing may provide a new gene therapeutic targeting approach based on CD44 pre-mRNA processing in vivo. This strategy incorporates utilization of CD44 alternative splicing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT) approach for the treatment of brain metastases from tumors of systemic origin. As initial steps towards the development of a gene therapeutic approach based on targeting tumor cell expression of specific CD44v alternatively spliced isoforms, we have: (1) developed a novel in vivo assay system that allows the rapid analyses of potentially therapeutic CD44 alternative splicing minigene constructs; and (2) cloned the E. coli cytosine deaminase (CD) gene and fused its enzymatically active domain to alternatively spliced CD44 exons (CD44/CD). Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated in E. coli cell lysates to be equal to that of wild type cytosine deaminase.

Alternative Splicing↗

PubMed2005

SpliceInfo: an information repository for mRNA alternative splicing in human genome.

We have developed an information repository named SpliceInfo to collect the occurrences of the four major alternative-splicing (AS) modes in human genome; these include exon skipping, 5'-alternative splicing, 3'-alternative splicing and intron retention. The dataset is derived by comparing the nucleotide and protein sequences available for a given gene for evidence of AS. Additional features such as the tissue specificity of the mRNA, the protein domain contained by exons, the GC-ratio of exons, the repeats contained within the exons, and the Gene Ontology are annotated computationally for each exonic region that is alternatively spliced. Motivated by a previous investigation of AS-related motifs such as exonic splicing enhancer and exonic splicing silencer, this resource also provides a means of identifying motifs candidates and this should help to identify potential regulatory mechanisms within a particular exonic sequence set and its two flanking intronic sequence sets. This is carried out using motif discovery tools to identify motif candidates related to alternative splicing regulation and together with a secondary structure prediction tool, will help in the identification of the structural properties of such regulatory motifs. The integrated resource is now available on http://SpliceInfo.mbc.NCTU.edu.tw/.

Alternative Splicing↗

PubMed2005

Regulation of alternative splicing of caspase-2 through an intracellular signaling pathway in response to pro-apoptotic stimuli.

Alternative splicing is an important mechanism in the generation of functionally distinct products from the same gene. Some apoptosis-regulating genes also undergo alternative splicing, generating splice variants that antagonzie normal transcripts on apoptosis. For example, caspase-2 is alternatively spliced, leading to exon 9-lacking caspase-2L (proapoptotic) and exon 9-containing caspase-2S (antiapoptotic) transcripts. Serine-arginine splicing factor proteins (SR proteins) are highly conserved and required for constitutive and alternative messenger RNA (mRNA) splicing. Their activity is regulated by reversible phosphorylation on serine residue. During apoptosis, many functional molecules undergo posttranslational modification, including phosphorylation, dephosphorylation, and caspase cleavage. In this study, we investigated the effect of proapoptotic stimuli on alternative splicing of caspase-2 mRNA in U937 cells. U937 cells were simulated with etoposide, staurosporine, pacritaxel, or cyclohexamide. We analzyed the alternative splicing of caspase-2 mRNA using reverse transcription-polymerase chain reaction. Etoposide, staurosporine, pacritaxel, and cyclohexamide treatment promoted exon-9 inclusion, increasing the ratio of caspase-2S to caspase-2L in a time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase-1, blocked etoposide-induced alternative splicing of caspase-2 mRNA. Furthermore, pretreatment of U937 cells with fumonisin B1, an inhibitor of ceramide synthase, also blocked alternative splicing of caspase-2 mRNA. These data demonstrate that endogenous ceramide generation and subsequent phosphatase activation during apoptosis are key steps in the alternative splicing of caspase-2 mRNA and further suggest a link between the signal-transduction pathway and alternative splicing.

Alternative Splicing↗

PubMed1996

Identification and characterization of two alternative splice variants of human interleukin-2.

Our previous work showed that alternative splicing is used to make an inhibitory variant of human interleukin (IL)-4. Because of homology between IL-4 and IL-2 proteins and receptors, we tested whether alternative splicing is used to generate similar inhibitory variants of human IL-2. Messenger RNA from peripheral blood mononuclear cells was subjected to reverse transcription-polymerase chain reaction using IL-2 exon 1- and exon 4-specific primers. Two amplification products, named IL-2delta2 and IL-2delta3, were found in addition to the native IL-2 product. The IL-2delta2 cDNA sequence was identical to IL-2 cDNA throughout the entire coding region, except exon 2 was omitted by alternative splicing. In IL-2delta3 cDNA, the third exon of IL-2 was omitted by alternative splicing. Unlike IL-2, IL-2delta2 and IL-2delta3 did not stimulate T cell proliferation. However, both inhibited IL-2 costimulation of T cell proliferation, and both inhibited cellular binding of rhIL-2 to high affinity IL-2 receptors. Thus, IL-2 is the second cytokine that uses alternative splicing to generate variants that are competitive inhibitors.

Alternative Splicing↗

PubMed2002

Selecting for functional alternative splices in ESTs.

The expressed sequence tag (EST) collection in dbEST provides an extensive resource for detecting alternative splicing on a genomic scale. Using genomically aligned ESTs, a computational tool (TAP) was used to identify alternative splice patterns for 6400 known human genes from the RefSeq database. With sufficient EST coverage, one or more alternatively spliced forms could be detected for nearly all genes examined. To identify high (>95%) confidence observations of alternative splicing, splice variants were clustered on the basis of having mutually exclusive structures, and sample statistics were then applied. Through this selection, alternative splices expected at a frequency of >5% within their respective clusters were seen for only 17%-28% of genes. Although intron retention events (potentially unspliced messages) had been seen for 36% of the genes overall, the same statistical selection yielded reliable cases of intron retention for <5% of genes. For high-confidence alternative splices in the human ESTs, we also noted significantly higher rates both of cross-species conservation in mouse ESTs and of validation in the GenBank mRNA collection. We suggest quantitative analytical approaches such as these can aid in selecting useful targets for further experimental characterization and in so doing may help elucidate the mechanisms and biological implications of alternative splicing.

Alternative Splicing↗