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Results for “Alternative Splicing”

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PubMed1999

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.

Alternative Splicing↗

PubMed2004

[Multidrug resistant effect of alternative splicing form of MAD2 gene-MAD2beta on human gastric cancer cell].

OBJECTIVE: To study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901. METHODS: RNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS. RESULTS: A fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells. CONCLUSION: MAD2beta could increase the multidrug resistance of SGC7901 cell line.

Adenocarcinoma↗

PubMed1996

Neuronal nitric-oxide synthase-mu, an alternatively spliced isoform expressed in differentiated skeletal muscle.

Nitric oxide (NO) functions as a molecular mediator in numerous processes in cellular development and physiology. Differential expression and regulation of a family of three NO synthase (NOS) gene products help achieve this diversity of action. Previous studies identify post-translational modification and interaction of NOS with specific protein targets as tissue-specific modes of regulation. Here, we show that alternative splicing specifically regulates neuronal NOS (nNOS, type I) in striated muscle. nNOS in skeletal muscle is slightly more massive than nNOS from brain owing to a 102-base pair (34-amino acid) alternatively spliced segment between exons 16 and 17. Following purification, this novel nNOS mu isoform has similar catalytic activity to that of nNOS expressed in cerebellum. nNOS mu appears to function exclusively in differentiated muscle as its expression occurs coincidentally with myotube fusion in culture. An isoform-specific antibody detects nNOS mu protein only in skeletal muscle and heart. This study identifies alternative splicing as a means for tissue-specific regulation of nNOS and reports the first additional protein sequence for a mammalian NOS since the original cloning of the gene family.

Alternative Splicing↗

PubMed1997

Alternatively spliced variants: a new view of the integrin cytoplasmic domain.

A large number of studies have underscored a major role for the integrin alpha beta cytoplasmic domains in the modulation of cell functions. Cytoplasmic domain variants of the beta 1, beta 3, beta 4, alpha 3, alpha 6 and alpha 7 subunits have been described. These molecules are generated by alternative splicing events and are expressed in a cell- or tissue-type specific manner. Some of these variants (beta 1C, beta 1D, alpha 6A and alpha 7A) are predominantly expressed upon differentiation and have been shown to be regulated during development. The studies on the structure-function relationship of the integrin variant subunits, published between 1989 and now, will be reviewed here for the first time. The results demonstrate that differences in the cytoplasmic domain do not affect either the alpha beta heterodimer formation or the ligand specificity. Instead, alternatively spliced integrin cytoplasmic domains appear to be essential modulators of receptor localization, cell proliferation and migration, as well as phosphorylation of signaling molecules. These observations lead to the current hypothesis that cell-type specific regulation of alternatively spliced integrin cytoplasmic domains may provide a highly specialized mechanism to control cell growth and intracellular signaling pathways.

Alternative Splicing↗

PubMed2004

SIAH1 targets the alternative splicing factor T-STAR for degradation by the proteasome.

T-STAR is one of three members of the SAM68 family of RNA-binding proteins that have been shown to be involved in various gene expression pathways including the control of pre-mRNA splicing. We employed a two-hybrid screen to identify proteins that interact with human T-STAR. The predominant interacting proteins were the E3 ubiquitin ligases SIAH1 and SIAH2. We found that SIAH1 bound to an octapeptide sequence in T-STAR targeting it for proteasome-dependent degradation. Rodent T-STAR orthologues (also known as etoile or SLM2) were not targeted for degradation by SIAH1. However a double amino acid substitution of mouse T-STAR that mimics the human SIAH1-binding site brought mouse T-STAR under in vivo control of SIAH1. Using a minigene transfection assay for alternative splicing activity we showed that human T-STAR, like its rodent orthologues can influence splice site choice and that human, but not mouse, T-STAR-dependent alternative splicing is modulated by SIAH1. Western blots of protein from purified germ cells indicated that SIAH1 protein expression peaks in meiosis. In mouse, T-STAR is co-expressed with SIAH1 during meiosis but, in humans, T-STAR is only strongly expressed after meiosis. Comparative sequence analysis showed SIAH-mediated proteasomal degradation of T-STAR has evolved in the primate lineage. Collectively these data suggest that SIAH-mediated down regulation of alternative splicing may be an important developmental difference between otherwise highly conserved T-STAR proteins.

Alternative Splicing↗

PubMed1990

Regulation of alternative splicing in the generation of isoforms of the mouse Ly-5 (CD45) glycoprotein.

Alternative splicing generates various Ly-5 glycoprotein isoforms of the cell surface that typify different cell lineages and stages of hematopoietic differentiation in the mouse; exons 4-6 are incorporated to generate a B-cell isoform (B220) and excluded from a T-cell isoform (T200), the other coding exons (3 and 7-33) being shared. As a first step to understanding the mechanisms regulating Ly-5 alternative splicing, and thus determining Ly-5 isoforms, a minigene representing exons 3-7 was transfected into Ly-5-expressor T cells and B cells and into nonexpressor L cells for comparison of splicing patterns. We conclude that all the information required for faithful splice-site selection according to cell type is contained within the resulting pre-mRNA. The splicing pattern manifested by nonexpressor L cells may represent a default and nonregulated type. We postulate trans-acting factor(s) to account for the selection of appropriate exons, and we provide support for this interpretation from analysis of fused hybrid T-B cells, which exhibited B-cell specific Ly-5 transcripts. Splicing patterns were well conserved despite substantial disruption of constructs. However, extensive deletion analyses suggested that cis sequences flanking and within exon 6 affect the exclusion of that exon in T cells.

Animals↗

PubMed2004

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue.

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3 , and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Alternative Splicing↗

PubMed2002

Mutations in the alternatively spliced exons of USH1C cause non-syndromic recessive deafness.

We have recently shown that USH1C underlies Usher syndrome type 1c (USH1C), an USH1 subtype characterized by profound deafness, retinitis pigmentosa, and vestibular dysfunction. USH1C encodes a PDZ-domain-containing protein, harmonin. Eight different Ush1c transcripts were identified in the mouse inner ear. Moreover, transcripts containing seven alternatively spliced exons (A-F, G/G) were found to be expressed in the inner ear, but not in the eye. These findings suggested that mutations involving USH1C might also be the cause of DFNB18, a form of non-syndromic deafness, which maps to a chromosomal region that includes USH1C. We screened 32 Chinese multiplex families with non-syndromic recessive deafness for USH1C mutations. In one family, congenital profound deafness without RP was associated with a C to G transversion in the alternatively spliced exon D, predicting an arginine to proline substitution at codon 608 in the proline-, serine- and threonine-rich region of harmonin. We also screened 320 deaf probands from other ethnic background and found three who were heterozygous for changes in the alternately spliced exons including Gly431Val in exon B, Arg620Leu and Arg636Cys in exon D. None of these mutations were detected in DNA from 200 control subjects with normal hearing including 110 Chinese. We also screened 121 non-Acadian probands with type 1 Usher syndrome. None carried any mutations in these exons of USH1C. Our findings show that USH1C mutations can also cause non-syndromic deafness and that some harmonin isoforms are specifically required for inner ear function.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Alternative splicing of human G6PD messenger RNA in K562 cells but not in cultured erythroblasts.

The biochemical properties of glucose-6-phosphate dehydrogenase (G6PD) vary in different tissues, and different protein isoforms of the enzyme have been described. Alternative splicing of G6PD intron VII has been detected in transformed lymphoblasts, granulocytes and spermatocytes; the function of this mRNA species is still unknown. We developed a PCR for detecting alternatively spliced G6PD mRNA in K562 and in erythroblasts at different stage of maturation obtained from human peripheral BFU-E in order to evaluate a possible physiological role during erythroid maturation. Trace events of alternative splicing of G6PD intron VII sequences were observed in K562 cells but not in BFU-E-derived erythroid precursors; we consider this phenomenon a non-functional activity in the cells analysed.

Base Sequence↗

PubMed1990

Expression of beta amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR.

We have analyzed alternatively spliced beta amyloid protein precursor (beta APP) mRNAs by using the polymerase chain reaction to amplify beta APP cDNAs produced by reverse transcription. With this approach the three previously characterized beta APP mRNAs (beta APP695, beta APP751, and beta APP770) are readily detected and compared in RNA samples extracted from specimens as small as a single cryostat section. We show that the results obtained with this method are not affected by partial RNA degradation and use it to identify a novel alternatively spliced beta APP714 mRNA that is present at low abundance in each of the many human brain regions, peripheral tissues, and cell lines that we have examined; demonstrate that nonneuronal cells in the adult human brain and meninges produce appreciable beta APP695, beta APP751, and beta APP770 mRNA; and identify changes in beta APP gene expression in the AD brain and meninges that may contribute to amyloid deposition.

Alzheimer Disease↗

PubMed2000

Expression of alternatively spliced FGF-2 antisense RNA transcripts in the central nervous system: regulation of FGF-2 mRNA translation.

The fibroblast growth factor-2 (FGF-2) gene is bidirectionally transcribed to produce the FGF-2 mRNA and a 1.5 kb antisense (FGF-AS) transcript complementary to the 3' untranslated region of the FGF-2 transcript. The FGF-AS RNA has been postulated to play a role in the post-transcriptional regulation of FGF-2, but this function has not been conclusively demonstrated. We characterized FGF-AS cDNAs from rat brain and C6 glioma cells, and investigated their role in regulation of FGF-2 expression. Three FGF-AS cDNAs were isolated; the full-length FGF-AS mRNA and two alternative splice variants lacking exon 2 or exons 2 and 3 of the FGF-AS sequence. The alternatively spliced FGF-AS RNAs are widely expressed in the CNS, whereas liver predominantly expressed the full-length transcript. The full-length and first splice variant encode 35 and 28 kDa isoforms of GFG, a MutT-related nuclear protein, whereas the second splice variant was not translated. The effect of FGF-AS RNA on FGF-2 expression was evaluated in stable C6 transfectants over-expressing the full-length or alternatively spliced FGF-AS RNA forms. All three constructs suppressed cellular FGF-2 protein (but not FGF-2 mRNA) levels, and this effect correlated directly with the level of FGF-AS RNA. Cellular FGF receptor content was increased and cell proliferation inhibited compared to wild type or vector-transfected cells, indicating disruption of the FGF-2 autocrine pathway by FGF-AS RNA. These findings demonstrate for the first time that the FGF-AS RNA regulates FGF-2 expression in mammalian cells, and suggest that this effect is exerted predominantly at the level of translation.

Alternative Splicing↗

PubMed2000

Expression of alternatively spliced FGF-2 antisense RNA transcripts in the central nervous system: regulation of FGF-2 mRNA translation.

Alternative Splicing↗

PubMed1992

An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.

Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.

Amino Acid Sequence↗

PubMed2006

Alpha-synuclein structure, posttranslational modification and alternative splicing as aggregation enhancers.

Alpha-synuclein aggregation is thought to be a key event in the pathogenesis of synucleinopathies. Although different alpha-synuclein alterations and modifications have been proposed to be responsible for early aggregation steps, the mechanisms underlying these events remain unclarified. Alpha-synuclein is a small protein localized to synaptic terminals and its intrinsic structure has been claimed to be an important factor for self-oligomerization and self-aggregation. Alpha-synuclein expression studies in cell cultures have demonstrated that posttranslational modifications, such as phosphorylation, oxidation, and sumoylation, are primarily involved in alpha-synuclein aggregation. Furthermore, in the last few years accumulating evidence has pointed to alternative splicing as a crucial mechanism in the development of neurodegenerative disorders. At least three different alpha-synuclein isoforms have been described as products of alternative splicing. Two of these isoforms (alpha-synuclein 112 and alpha-synuclein 126) are shorter proteins with probably altered functions and aggregation propensity. The present review attempts to summarize the data so far available on alpha-synuclein structure, posttranslational modifications, and alternative splicing as possible enhancers of aggregation.

Alternative Splicing↗

PubMed1991

Alternative splicing of fibronectin: three variants, three functions.

Fibronectin (FN) is a multi-functional extracellular matrix protein required for cell adhesion and migration, blood clotting, wound healing, and oncogenic transformation. The functional complexity is paralleled by structural diversity in that multiple forms of FN are generated by cell type-specific alternative splicing. In the rat, up to 12 different combinations of the three alternatively spliced segments (EIIIA, EIIIB, and the V region) are produced. What effects do these segments have on FN function? Recently, progress has been made in the identification of specific activities for the three variants of the V region, V120, V95, and V0. FN-mediated cell adhesion, FN synthesis and secretion, and incorporation into blood clots are differentially affected by these isoforms. These results suggest that cellular behavior is modulated by environmental cues provided by different types and proportions of alternatively spliced FN variants.

Animals↗

PubMed1991

Demonstration of the existence of the alternatively spliced form of thyroid peroxidase in normal thyroid.

We have investigated whether the alternatively spliced form of thyroid peroxidase (TPO), which has been shown clearly to occur in Graves' thyroid, is present in normal thyroid. We have performed a polymerase chain reaction on cDNAs prepared from normal thyroid mRNAs using primers 100 bases up-stream and down-stream from the 171 nucleotides that have been shown to be spliced out. Size analysis of the polymerase chain reaction products by agarose gel electrophoresis revealed bands at 0.2 and 0.37 kilobases (kb), sizes predicted to be obtained when the two forms of TPO cDNA are present. This was confirmed by hybridization with a 32P-labeled probe corresponding to the 20 nucleotides of the junction site. After stringent washing, the 0.2-kb band gave a clear positive signal, which was stronger than that of the 0.37-kb band despite the latter cDNA being more abundant. This demonstrates clearly that the alternatively spliced form of TPO exists in normal thyroid. Its significance is discussed.

Base Sequence↗

PubMed1995

Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing.

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

Alternative Splicing↗

PubMed1995

APC mutation in the alternatively spliced region of exon 9 associated with late onset familial adenomatous polyposis.

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.

Adenoma↗