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Results for “Alternative Splicing”

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PubMed1998

Identification of alternatively spliced Na(+)-Ca2+ exchanger isoforms expressed in the heart.

Alternatively spliced isoforms of Na(+)-Ca2+ exchanger were found from various tissues and species. RT-PCR amplification was performed on the basis of our cloned mouse cardiac Na(+)-Ca2+ exchanger and four alternatively spliced isoforms of Na(+)-Ca2+ exchanger were identified. Three (NCX1.3, NCX1.4, and NCX1.12) of them were first identified in the heart, and one isoform (NCX1.12) was a novel spliced variant. These four spliced variants were present in the embryonic and adult atria and ventricles. Different cell types of the heart expressed different spliced isoforms of Na(+)-Ca2+ exchanger. Southern blot analysis indicated that the Na(+)-Ca2+ exchanger gene existed as a single copy in the mouse genome. Thus, the Na(+)-Ca2+ exchanger isoforms expressed in mouse heart are consistent with being produced by alternative splicing and they may have different functions in various cell types in the mouse heart.

Alternative Splicing↗

PubMed1993

Quantitative analysis of alternative splicing options of human plasma membrane calcium pump genes.

The alternative splicing options and the quantitative tissue distribution of the transcripts of the four currently known human plasma membrane calcium pump (PMCA) genes have been analyzed in seven tissues (cerebral cortex, skeletal and heart muscle, stomach, liver, lung, and kidney) by quantitative polymerase chain reaction on reverse transcribed mRNA with glyceraldehyde-3-phosphate dehydrogenase as the internal standard. The mRNAs of genes 1 and 4 were found to be present in similar amounts in all tissues, whereas the transcripts of genes 2 and 3 were expressed in a tissue-specific manner, i.e. their amounts were highest in fetal skeletal muscle and brain. Alternative splicing was found to occur in the PMCA transcripts at two major regulatory sites (sites A and C), adjacent to the amino-terminal phospholipid-responsive region and within the carboxyl-terminal calmodulin binding domain, respectively. Novel splicing variants not described previously for human genes were detected for hPMCA3 and 4 at site A and for hPMCA1, 2, and 3 at site C. For all genes a common splice variant was found at both splice sites. The common splice variant at site A was characterized by the inclusion of a small exon (hPMCA1, 39 base pairs (bp); hPMCA2, 42 bp; hPMCA3, 42 bp; hPMCA4, 36 bp). In the common splice variant at site C, an exon (hPMCA1, 154 bp; hPMCA2, 227 bp; hPMCA3, 154 bp; hPMCA4, 178 bp) was excluded in the mRNA. All genes normally express these main splice variants in all tissues in which the corresponding isoform is present. The splicing complexity at site C was found to be augmented in the transcripts of PMCA2 and PMCA3 through the use of additional exons, and in PMCA1 and 3 through the use of additional internal splice sites in the single alternatively spliced 154-base pair exon.

Aged↗

PubMed2003

Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences.

We report here a genome-wide analysis of alternative splicing in 2 million human expressed sequence tags (ESTs), to identify splice forms that are up-regulated in tumors relative to normal tissues. We found strong evidence (P < 0.01) of cancer-specific splice variants in 316 human genes. In total, 78% of the cancer-specific splice forms we detected are confirmed by human-curated mRNA sequences, indicating that our results are not due to random mis-splicing in tumors; 73% of the genes showed the same cancer-specific splicing changes in tissue-matched tumor versus normal datasets, indicating that the vast majority of these changes are associated with tumorigenesis, not tissue specificity. We have confirmed our EST results in an independent set of experimental data provided by human-curated mRNAs (P-value 10(-5.7)). Moreover, the majority of the genes we detected have functions associated with cancer (P-value 0.0007), suggesting that their altered splicing may play a functional role in cancer. Analysis of the types of cancer-specific splicing shifts suggests that many of these shifts act by disrupting a tumor suppressor function. Sur prisingly, our data show that for a large number (190 in this study) of cancer-associated genes cloned originally from tumors, there exists a previously uncharacterized splice form of the gene that appears to be predominant in normal tissue.

Alternative Splicing↗

PubMed2001

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated.

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.

Alternative Splicing↗

PubMed1996

Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer.

Alternative Splicing↗

PubMed1992

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.

Amino Acid Sequence↗

PubMed1991

The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors.

The reverse transcription-polymerase chain reaction was used to examine alternative splicing at each of the three fibronectin exons known to undergo alternative splicing, i.e. extra domain A (ED-A), extra domain B (ED-B), and type III connecting sequence (IIICS). Ratios of fibronectin mRNAs with or without a given exon were determined in several rat tissues and human cell lines during aging in vivo and cellular senescence in vitro. We demonstrate that statistically significant shifts in the alternative splicing of fibronectin occur during aging in vivo and in vitro. Since all three alternatively spliced exons are spliced out at a higher frequency in aging tissues and cells, the fibronectin protein produced by old cells should be slightly smaller than that obtained from young cells. The reverse transcription-polymerase chain reaction demonstrates tissue-specific patterns of alternative splicing in several tissues. Whereas fibronectin mRNAs from adult rat tissues were found to range from 0 to 25% ED-A+ and from 0 to 10% ED-B+, fibronectin mRNAs from cultured cell lines were found to be approximately 50-60% ED-A+ and 15-25% ED-B+. We observed similarity in splicing of fibronectin RNA by the different cultured cell lines obtained from many tissues and attribute this observation to the effect of growth factors. We demonstrate that serum deprivation; placement of cells into primary culture; and growth factors such as transforming growth factor beta 1, retinoic acid, and 1,25-dihydroxyvitamin D3 can all change the alternative splicing of fibronectin pre-mRNA in the ED-A, ED-B, and type III connecting sequence exons. Possible mechanisms for the regulation of the alternative splicing of fibronectin RNA by growth factors are discussed.

Aging↗

PubMed2005

Candidates for tumor-specific alternative splicing.

Gene expression can be regulated not only by transcription and post-transcriptional modifications, but also by splicing regulation. Recent genome-wide analyses have indicated that up to 70% of human genes may have alternatively spliced forms, suggesting that splicing regulation affects a wide range of gene expression. Tumor tissues show significantly altered protein expressions, and this is also thought to be affected by alternative splicing. Although some alternative splicing events have been reported to be cancer specific and others have been predicted from database analyses, the process of alternative splicing and its regulatory machinery are hardly understood. We searched for and detected alternative splicing events that alter protein splicing in all or a subset of tumor tissues. The results revealed tissue-specific alterations of splicing regulation by tumorigenesis, and regulatory cis-element analyses further suggested that multiple splicing regulatory machineries were affected by this process.

Alternative Splicing↗

PubMed1999

Alternative splicing and programmed cell death.

Programmed cell death (PCD) is critical for development and homeostasis of multicellular organisms. Genetic and biochemical studies have revealed that PCD is under complex and delicate regulation. An important level of such regulation may be pre-mRNA splicing as suggested by the observation that a number of PCD regulatory genes are expressed as functionally distinct or even antagonistic isoforms as a result of alternative splicing. Studies on alternative splicing of these genes are reviewed here. Expression and function of a large number of genes involved in PCD are regulated by alternative splicing, including death receptors and intracellular components of the death machinery. Alternative splicing affects not only intracellular distribution but also functional activity of these death regulators, providing a fine-tuning mechanism in modulating a presumably tightly controlled process of cell death.

Alternative Splicing↗

PubMed1997

Induction of alternative splicing of HLA-B27 by bacterial invasion.

OBJECTIVE: Alternative splicing of certain class I major histocompatibility complex pre-messenger RNA (pre-mRNA) is known to lead to generation of a cell-free soluble protein analog. This study was undertaken to examine whether this process occurs with HLA-B27, whether the process is modified by arthritis-causing bacteria, and whether the assembly of the soluble molecules follows the same pathway as the integral parent molecules. METHODS: Alternative splicing of pre-mRNA was analyzed by reverse transcriptase-polymerase chain reaction, and assembly of soluble HLA-B27 by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. RESULTS: There was alternative splicing of the pre-mRNA of HLA-B27. The process could be amplified by invasion with Salmonella or Yersinia bacteria. The soluble HLA-B27 was assembled in a pathway similar to that of the parent molecule. CONCLUSION: The association between arthritis-causing bacteria and HLA-B27 positive cells is a complex event. Soluble HLA-B27 is a potential key player.

Alternative Splicing↗

PubMed2005

ECgene: genome annotation for alternative splicing.

ECgene provides annotation for gene structure, function and expression, taking alternative splicing events into consideration. The gene-modeling algorithm combines the genome-based expressed sequence tag (EST) clustering and graph-theoretic transcript assembly procedures. The website provides several viewers and applications that have many unique features useful for the analysis of the transcript structure and gene expression. The summary viewer shows the gene summary and the essence of other annotation programs. The genome browser and the transcript viewer are available for comparing the gene structure of splice variants. Changes in the functional domains by alternative splicing can be seen at a glance in the transcript viewer. We also provide two unique ways of analyzing gene expression. The SAGE tags deduced from the assembled transcripts are used to delineate quantitative expression patterns from SAGE libraries available publically. Furthermore, the cDNA libraries of EST sequences in each cluster are used to infer qualitative expression patterns. It should be noted that the ECgene website provides annotation for the whole transcriptome, not just the alternatively spliced genes. Currently, ECgene supports the human, mouse and rat genomes. The ECgene suite of tools and programs is available at http://genome.ewha.ac.kr/ECgene/.

Algorithms↗

PubMed2005

Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing.

During pre-mRNA splicing, introns are removed and exons are ligated to form an mRNA. Exon choice is determined by different nuclear protein concentrations varying among tissues and cell types or by developmental stage. These can be altered by different cellular circumstances such as physiological stimuli, environmental effects and phosphorylation state. The splicing factor hSlu7 plays an important role in 3' splice site selection during the second step of splicing in vitro and has been suggested to affect alternative splicing in vivo. Our results indicate that an ultraviolet-C (UV-C) stress stimulus triggers changes in the alternative splicing patterns of cellular genes by decreasing the nuclear concentration of hSlu7 through the modulation of its nucleus-to-cytoplasm transport. This shift is mostly dependent on the Jun N-terminal kinase (JNK) cascade. Although we found by RNAi knockdown that hSlu7 is not essential for cell viability, its nuclear concentration effects exon choice and inclusion:skipping ratio of alternative splicing. A possible spatial and temporal regulatory mechanism by which hSlu7 protein levels are regulated within the nucleus is suggested, thus implying a broad effect of hSlu7 on alternative splicing.

Active Transport, Cell Nucleus↗

PubMed1991

At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are expressed during rat heart development.

The major membrane-associated or transmembrane isoforms of the neural cell adhesion molecule (NCAM) are generated by alternative splicing at the 3' end of the mRNA. Further diversity in NCAM structure is observed in the extracellular region of the polypeptide, where the insertion of additional amino acid residues can result from alternative splicing events occurring at the exon 7-exon 8 and exon 12-exon 13 junctions. Here we report the characterization of tissue-specific patterns of alternative splicing at the exon 12-exon 13 junction by using the polymerase chain reaction. Nine alternatively spliced sequences in rat heart between exon 12 and exon 13 were identified. Each sequence consisted of different combinations of the three small exons (15, 48, and 42 bp in length) and the AAG triplet that make up MSD1, the 108-bp muscle-specific sequence found in human skeletal muscle NCAM (G. Dickson, H.J. Gower, C. H. Barton, H. M. Prentice, V. L. Elsom, S. E. Moore, R. D. Cox, C. Quinn, W. Putt, and F. S. Walsh, Cell 50:1119-1130, 1987). Although the rat equivalent of MSD1 (designated 15+ 48+ 42+ 3+) was detected in all ages of heart examined, it was only one of four or five major splice combinations at any given age. The only alternatively spliced sequence found in the exon 7-exon 8 junction of heart NCAM mRNA was the 30-bp variable alternatively spliced exon previously identified in rat brain. Twenty-seven NCAM forms with distinct sequences were found by analysis of individual NCAM transcripts from postnatal day 1 heart tissue for alternative splicing at the exon 7-exon 8 junction, the exon 12-exon 13 junction and the 3' end. Several combinations of splicing patterns in these three different regions of the gene appeared to be preferentially expressed. The observation that the expression of alternatively spliced forms of NCAM is developmentally regulated suggests a role for NCAM diversity in cardiac development.

Amino Acid Sequence↗

PubMed2006

Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration.

Mitochondrial damage is linked to many neurodegenerative conditions, such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. These diseases are associated with changes in the splicing pattern of individual mRNAs. Here, we tested the hypothesis that mitochondrial damage modulates alternative splicing, not only of a few mRNAs, but in a general manner. We incubated cultured human neuroblastoma cells with the chemical agent paraquat (a neurotoxin that interferes with mitochondrial function, causing energy deficit and oxidative stress) and analysed the splicing pattern of 13 genes by RT-PCR. For all mRNAs that are alternatively spliced, we observed a dose- and time-dependent increase of the smaller isoforms. In contrast, splicing of all constitutive splicing exons that we monitored did not change. Using other drugs, we show that the modulation of alternative splicing correlates with ATP depletion, not with oxidative stress. Such drastic changes in alternative splicing are not observed in cell lines of non-neuronal origin, suggesting a selective susceptibility of neuronal cells to modulation of splicing. As a significant percentage of all mammalian mRNAs undergo alternative splicing, we predict that mitochondrial failure will unbalance a vast number of isoform equilibriums, which would give an important contribution to neurodegeneration.

Adenosine Triphosphate↗

PubMed2006

Analysis of alternatively spliced isoforms of human LDL receptor mRNA.

BACKGROUND: The low density lipoprotein receptor (LDLR) family is a family of structurally related cell surface receptors with conserved exon/intron organization. Several members of this family have been shown to undergo alternative splicing. However, no alternative splicing of the LDLR pre-mRNA has so far been described. METHODS: In the present study alternative splicing of human LDLR pre-mRNA has been studied in eight different tissues and four different cell lines using reverse transcription (RT) PCR. A quantitative real-time PCR with exon-exon boundary spanning primers was established to measure the relative amount of two novel isoforms. RESULTS: Several novel isoforms were identified by RT-PCR of which the isoforms lacking exon 4 or 12 were two of the most prominent. Although highly detectable by RT-PCR, the quantification by real-time PCR revealed low levels of these isoforms. CONCLUSIONS: Novel isoforms of LDLR mRNA are described. Quantification by real-time PCR of two of the alternatively spliced isoforms revealed low amount of these isoforms in the examined tissues and cell lines. Further investigations are needed to evaluate if these isoforms represent functional transcripts of LDLR mRNA.

Alternative Splicing↗

PubMed2001

Five novel alternatively spliced transcripts of DNA (cytosine-5) methyltransferase 2 in human peripheral blood leukocytes.

Alternative splicing of RNA molecules transcribed from DNA (cytosine-5) methyltransferases has been proposed as a mechanism by which methylation is able to effect diverse biological processes in higher eukaryotes. This study has investigated transcriptional versatility of DNA (cytosine-5) methyltransferase 2, which may methylate cytosine residues within 5'-CCTGG-3' pentanucleotides in regions of the human genome devoid of 5'-CG-3' methylation. Five novel splice variants of DNA (cytosine-5) methyltransferase 2 were identified in the peripheral blood leukocytes of healthy subjects following cloning and sequencing of RT-PCR products amplified using gene specific oligodeoxyribonucleotide primers. The generation of some of these splice variants may be influenced by the formation of secondary structures within pre-mRNA due to the repetition of sequences flanking alternatively spliced exons in a reverse and complementary orientation on the same strand. These findings enable novel approaches to investigate the role of RNA secondary structures in alternative splicing. The DNA (cytosine-5) methyltransferase 2 splice variants are generated in all the major cell types of peripheral blood, as well as in neoplastic lymphoid cells indicating that they are unlikely to generate proteins involved in control of the cell cycle or cellular differentiation. Interestingly, the gene products generated by some splice variants completely or partially lack highly conserved amino acid motifs shown to be important for the catalysis of cytosine methylation. The possibility cannot be excluded, therefore, that alternative splicing of DNA (cytosine-5) methyltransferase 2 pre-mRNA may generate protein isoforms which have different methylating capabilities or which are involved in biological processes other than the catalysis of cytosine methylation.

Alternative Splicing↗

PubMed2002

Alternative splicing of the adenylyl cyclase stimulatory G-protein G alpha(s) is regulated by SF2/ASF and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and involves the use of an unusual TG 3'-splice Site.

The factors involved in regulating alternative splicing of the human adenylyl cyclase stimulatory G-protein G alpha(s) in different cell types remain undefined. We have designed a G alpha(s) minigene that retains the signals required for G alpha(s) alternative splicing in vivo. Employing transient transfection of human myometrial smooth muscle cells and HeLa cells, as well as in vitro splicing assays, we have provided evidence that the antagonistic splicing factors SF2/ASF and hnRNPA1 act as potent regulators of G alpha(s) isoform expression in these cells. Both SF2/ASF and hnRNPA1 control the selection of competing 5'-splice sites and respectively promote inclusion or skipping of the small cassette-type exon 3 of G alpha(s) transcripts, resulting in the generation of G alpha(s)-long and G alpha(s)-short mRNA isoforms. We have also provided evidence that SF2/ASF and hnRNPA1 play a role in 3'-splice site selection involving the use of a non-canonical TG 3'-splice site preceding exon 4. Using a score-matrix analysis to identify putative exonic enhancer sequences (ESEs), we found multiple high score ESE motifs for SF2/ASF, SC35, and SRp40 in exon 3 of G alpha(s). These results suggest that tissue-specific expression of SF2/ASF and hnRNPA1 governs the expression of alternative isoforms of G alpha(s) in these different cells types.

Adenylyl Cyclases↗

PubMed2005

Cardiac tissue-specific repression of CELF activity disrupts alternative splicing and causes cardiomyopathy.

Members of the CELF family of RNA binding proteins have been implicated in alternative splicing regulation in developing heart. Transgenic mice that express a nuclear dominant-negative CELF protein specifically in the heart (MHC-CELFDelta) develop cardiac hypertrophy and dilated cardiomyopathy with defects in alternative splicing beginning as early as 3 weeks after birth. MHC-CELFDelta mice exhibit extensive cardiac fibrosis, severe cardiac dysfunction, and premature death. Interestingly, the penetrance of the phenotype is greater in females than in males despite similar levels of dominant-negative expression, suggesting that there is sex-specific modulation of splicing activity. The cardiac defects in MHC-CELFdelta mice are directly attributable to reduced levels of CELF activity, as crossing these mice with mice overexpressing CUG-BP1, a wild-type CELF protein, rescues defects in alternative splicing, the severity and incidence of cardiac hypertrophy, and survival. We conclude that CELF protein activity is required for normal alternative splicing in the heart in vivo and that normal CELF-mediated alternative splicing regulation is in turn required for normal cardiac function.

Alternative Splicing↗