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At least 595 records · Page 33Linked to original sources
PubMed1994

Cloning and characterization of alternatively spliced isoforms of rat tenascin. Platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells.

To understand the alteration of extracellular matrix composition evoked by chemotactic factors, we have studied the expression of adhesive (fibronectin) and anti-adhesive (tenascin) proteins in response to platelet-derived growth factor-BB (PDGF-BB), a potent chemoattractant for rat aortic smooth muscle cells (ASMC). PDGF-BB markedly induced two major tenascin mRNA transcripts, whereas fibronectin mRNA levels did not change. The results of immunoprecipitation studies paralleled Northern blot data. Since alternative splicing is responsible for the generation of multiple tenascin mRNAs in other cell types, we studied the effect of chemotactic factors on the relative abundance of tenascin isoforms. The alternatively spliced region of ASMC-derived rat tenascin was amplified and the identity of the products confirmed by sequencing. Three major polymerase chain reaction products were detected: a 1727-base pair unspliced form which was maximal at 2 h and 635- and 362-base pair products which were more abundant at 8 h after treatment with PDGF-BB or angiotensin II. Functional studies showed that the unspliced isoform of human tenascin inhibited attachment of both human and rat ASMC to fibronectin. These results suggest that PDGF-BB markedly up-regulates the expression of tenascin variants, which may lead to destabilization of cell-matrix interactions and promotion of cell migration.

Alternative Splicing↗
PubMedDate not supplied

[Analysis of the dynamics of transitions in Leptomonas collosoma spliced leader RNA and a possible role of gaps upon splicing].

In [1], the existence of two competing secondary structures at the 5'-end of Leptomonas collosoma spliced leader RNA was shown. On going from one steady-state secondary structure to the other, and vice versa, RNA passes through several states in which the chain gets tied up into nodes. If the transitions are reversible, the nodes are figurative both in the forth and back directions although the secondary structures that arise in this process are quite different, and the forth and back routs do not coincide. It is assumed that under certain conditions, the chain can get tied into the next node of series 5(2), which most likely will not get untied. This node is tightened around GG nucleotides. In this case, the migration of the gap is possible, as a result of which the chain can break. Independent experiments indicate that the RNA chain breaks just at this site upon splicing. Based on these results, a possible role of nodes in the enzymatic activity is discussed.

Animals↗
PubMedDate not supplied

Serum assay of soluble CD44 standard (sCD44-st), CD44 splice variant v5 (sCD44-v5), and CD44 splice variant v6 (sCD44-v6) in patients with epithelial ovarian cancer.

Different variants of the cell adhesion molecule CD44 have been involved in malignant transformation and cancer metastasis. In the present investigation we assessed the preoperative serum levels of soluble CD44 standard (sCD44-st), sCD44 splice variant 5 (sCD44-v5), and sCD44 splice variant 6 (sCD44-v6) in 51 patients with ovarian cancer. Median preoperative sCD44-st, sCD44v5, and sCD44-v6 levels were 417 ng/ml (range, 240- > 602 ng/ml), 78 ng/ml (range, 5-314 ng/ml), and 86 ng/ml (range, 1-243 ng/ml), respectively. No significant relationship was detected between sCD44-st concentrations and the common clinicopathological variables. Conversely, sCD44-v5 and sCD44-v6 levels were significantly lower in FIGO stage III-IV than in stage I disease (p < 0.0001 and p = 0.001, respectively). Moreover, with regard to advanced ovarian cancer, sCD44-v5 levels were lower in patients with poorly differentiated (G3) than in those with moderately (G2) or well (G1) differentiated tumors (p = 0.038), as well as in patients whose residual disease was > 2 cm than in those with smaller residuum (p = 0.025). Similarly, sCD44-v6 levels were lower in patients with large residual disease (p = 0.05). The median value of serum sCD44-v6 was lower in patients with G3 than in those with G1-G2 tumor, but the difference was not significant. In conclusion, sCD44-st, sCD44-v5, and sCD44-v6 are detectable in sera from patients with epithelial ovarian cancer. A reduction in preoperative sCD44-v5 and sCD44-v6 levels seems to be associated with advanced, poorly differentiated tumors and with large residual disease after first surgery, and it might reflect an increased biological aggressiveness of the malignancy.

Alternative Splicing↗
PubMed1999

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.

Animals↗
PubMed2003

Clinical heterogeneity and molecular findings in five Polish patients with glycerol kinase deficiency: investigation of two splice site mutations with computerized splice junction analysis and Xp21 gene-specific mRNA analysis.

Five cases of glycerol kinase deficiency are presented with clinical, biochemical, and genetic results. Two had the glycerol kinase deficiency as part of an Xp21 contiguous gene deletion syndrome-complex form-and three had an isolated form of the enzyme deficiency. In these we found two splice site mutations (IVS1+4A>G, IVS9-1G>T) and one insertion (1393_1394insG). In patients with the complex form, a deletion of the DAX1, GK genes and the distal part of the DMD gene was found. A computerized study was performed to predict the effects of the splice site mutations. It showed that the IVS9-1G>T mutation substantially altered and removed the wild-type site and enhanced a cryptic site seven nucleotides downstream, and that the IVS1+4A>G diminished the strength of the wild-type donor site from strong to leaky. To verify these predictions, we developed an RT-PCR system with gene-specific primers that exclusively amplifies the Xp21 glycerol kinase gene transcript. Identification of individuals at risk is motivated by a need to avoid delay in a correct diagnosis. For reliable identification of heterozygotes for isolated glycerol kinase deficiency, knowledge of the specific mutation in the proband is required. This is easily obtained with the RT-PCR analyses developed in this study.

Adrenal Insufficiency↗
PubMedDate not supplied

The serum assay of soluble CD44 standard (sCD44-st), CD44 splice variant 5 (sCD44-v5), and CD44 splice variant 6 (sCD44-v6) in patients with cervical cancer.

The pretreatment serum levels of soluble CD44 standard (sCD44st), CD44 splice variant 5 (sCD44-v5), and CD44 splice variant 6 (sCD44-v6) were retrospectively measured in 37 patients with untreated cervical cancer and in 36 patients with benign gynecological diseases as controls. Median sCD44-st levels were significantly higher in patients with cervical cancer than in controls (547 ng/ml, range 244-880 ng/ml versus 400.5 ng/ml, range 217-723 ng/ml, p = 0.004), whereas sCD44-v5 and sCD44-v6 concentrations were significantly lower in the former (34 ng/ml, range 0-140 ng/ml versus 44 ng/ml, range 11-109 ng/ml, p = 0.038; and 37 ng/ml, range 1-191 ng/ml versus 52.5 ng/ml, range 11-173 ng/ml, p = 0.007, respectively). sCD44-st, sCD44-v5, and sCD44-v6 levels were not related to FIGO stage and histologic type. Moreover, among patients with stage Ib-IIa cervical cancer, the preoperative levels of these glycoproteins correlated with neither the common prognostic variables nor the clinical outcome. Therefore, the serum assay of sCD44-st, sCD44-v5, and sCD44-v6 seems to have no clinical relevance for the management of patients with cervical cancer.

Adenocarcinoma↗
PubMed2006

Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides.

Antisense oligonucleotides (AONs) can be used to correct the disrupted reading frame of Duchenne muscular dystophy patients (DMD). We have a collection of 121 AONs, of which 79 are effective in inducing the specific skipping of 38 out of the 79 different DMD exons. All AONs are located within exons and were hypothesized to act by steric hindrance of serine-arginine rich (SR) protein binding to exonic splicing enhancer (ESE) sites. Indeed, retrospective in silico analysis of effective versus ineffective AONs revealed that the efficacy of AONs is correlated to the presence of putative ESE sites (as predicted by the ESEfinder and RESCUE-ESE software). ESE predicting software programs are thus valuable tools for the optimization of exon-internal antisense target sequences.

Dystrophin↗
PubMed1992

A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy.

Gyrate atrophy (GA) is an autosomal recessive eye disease involving a progressive loss of vision due to chorioretinal degeneration in which the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is defective. Two sisters with GA are described in this study in whom an A-to-G substitution at the 3' splice acceptor site of intron 4 in one allele of the OAT gene results in a truncated OAT mRNA devoid of exon 5 sequence. The mutation in the other allele was identified to be a mis-sense mutation at codon 318 by denaturing gradient gel electrophoresis and direct sequencing of the polymerase chain reaction (PCR)-amplified DNA. Thus, these GA patients are compound heterozygotes with respect to mutations in the OAT gene that result in inactivation of OAT.

Adenine↗
PubMed2004

Splicing alterations in human renal allografts: detection of a new splice variant of protein kinase Par1/Emk1 whose expression is associated with an increase of inflammation in protocol biopsies of transplanted patients.

Protein kinase Emk1/Par1 (GenBank accession no. X97630) has been identified as a regulator of the immune system homeostasis. Since immunological factors are critical for the development of chronic allograft nephropathy (CAN), we reasoned that expression of Par1/Emk1 could be altered in kidney allografts undergoing CAN. In this paper, we have analysed the association among renal allograft lesions and expression of Par1/Emk1, studied by RT-PCR on total RNA from 51 protocol biopsies of transplanted kidneys, five normal kidneys, and five dysfunctional allografts. The most significant result obtained has been the detection of alterations in the normal pattern of alternative splicing of the Par1/Emk1 transcript, alterations that included loss of expression of constitutively expressed isoforms, and the inclusion of a cryptic exon to generate a new Emk1 isoform (Emk1C). Expression of Emk1C was associated with an increase in the extension of the interstitial infiltrate (0.88+/-0.33 in Emk1C([+]) vs. 0.41+/-0.50 in Emk1C([-]); P<0.011), and with a trend to display higher interstitial scarring (0.66+/-0.70 vs. 0.29+/-0.52; P=0.09) in protocol biopsies when evaluated according to the Banff schema. Moreover, a higher mean arterial pressure (MAP) was also observed (110+/-11 vs. 99+/-11 mm Hg; P=0.012). From these results we propose that Par1/Emk1 could have a role in the development of CAN in kidney allografts.

Adult↗
PubMed2000

Human 18 kDa phosphotyrosine protein phosphatase (ACP1) polymorphism: studies of rare variants provide evidence that substitutions within or near alternatively spliced exons affect splicing result.

The mammalian low molecular weight phosphotyrosine protein phosphatase is expressed as two distinct isoforms. The human 'fast' and 'slow' isoforms differ only in the sequence of an internal segment of 34 residues, and the ACP1 gene contains two adjacent exons (E3F and E3S) which encode these segments. We have previously suggested that the fast and slow isoforms are generated by mutually exclusive pre-mRNA splicing of E3F and E3S. The common alleles ACP1*A, *B and *C express the fast and slow isoforms in different ratios. The *A and *C alleles differ from *B by C --> T transitions in E3S and E3F respectively. To test the idea that the fast : slow ratio is determined by nucleotide substitutions in the E3F-I3F-E3S region, four groups of rare ACP1 variants with unusual fast : slow ratios and the rare *E and *R alleles, expressing fast&ratio;slow ratios similar to *C and *B, respectively, were analysed. Gene segments of the I2-I3S region were amplified by PCR and analysed by SSCP and variant bands were excised and sequenced. For each of the rare isozymic variants one of six different nucleotide substitutions in E3F (nts+42, +85, +109, +110), I3F (nt+1) and I3S (nt+8) was observed. The *E and *R alleles showed C and B sequence, respectively, in accordance with the fast : slow ratio. The results support the hypothesis that the fast : slow ratio is constitutive.

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↗
PubMed2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia differentiation and determination of cardiac left-right asymmetry via regulation of Arl13b splicing.

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephaloph&#x14f;nus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

ARL13B↗
PubMed2005

Identification of four novel exon 5 splice variants of the mouse mu-opioid receptor gene: functional consequences of C-terminal splicing.

The rat mu-opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for mu-opioids in receptor-binding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of mu-agonists to each other varied from variant to variant in guanosine 5'-O-(3-[35S]thio)triphosphate-binding studies, as shown by morphine-6beta-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was mu-selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the mu-agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the mu-specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of mu-opioids.

Animals↗
PubMed1993

Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA.

The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.

Alternative Splicing↗
PubMed1992

Alternatively-spliced p53 mRNA in the FAA-HTC1 rat hepatoma cell line without the splice site mutations.

A novel mutation of the p53 gene has been found in a rat hepatoma cell line, FAA-HTC1. This cell line carried two kinds of abnormal p53 transcripts; one lacked the exon 8 sequence, and the other had a single base substitution G to T which resulted in a new stop codon in exon 8. In the genomic DNA, this base substitution in exon 8 was present, indicating that both transcripts were transcribed from the mutated gene. No mutation was detected in its two flanking introns. In this cell line, the exon-deleted transcript seems to be generated by exon skipping due to an unknown mechanism other than splice site mutations.

Animals↗
PubMed2004

The yeast splicing factor Prp40p contains functional leucine-rich nuclear export signals that are essential for splicing.

To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae. Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality. The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively. The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter. This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality. Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p. One of Prp40p's two NESs lies within the internal deletion. We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner. Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect. Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event.

Active Transport, Cell Nucleus↗
PubMed1992

Structure of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and characterization of alternative splicing and transcription products. Skeletal muscle-specific splicing results in a plasma membrane Ca(2+)-ATPase with a novel calmodulin-binding domain.

We have isolated the rat gene encoding isoform 3 of the plasma membrane Ca(2+)-ATPase (PMCA3) and have determined its exon/intron organization. The PMCA3 gene contains 24 exons and spans approximately 70 kilobases. In addition, we have analyzed the splicing and polyadenylation patterns leading to the production of an alternative 4.5-kilobase (PMCA3) skeletal muscle mRNA that differs from the previously characterized 7.5-kilobase brain mRNA (Greeb, J., and Shull, G. E. (1989) J. Biol. Chem. 264, 18569-18576). cDNA cloning, Northern blot hybridization, and polymerase chain reaction analyses of the 4.5-kilobase mRNA demonstrate (i) the inclusion of a novel 68-nucleotide exon (exon 22) that is specific for skeletal muscle and significantly alters the calmodulin-binding domain and (ii) the utilization of an alternative polyadenylation site following exon 23 which eliminates the last coding exon (exon 24) and 3'-untranslated sequence of the 7.5-kilobase mRNA. We have also identified a 42-nucleotide exon (exon 8) that is included in the skeletal muscle PMCA3 mRNAs, but may be either included or excluded in the brain mRNAs. Exon 8 is inserted immediately before the sequence encoding a putative phospholipid binding domain and thus may alter regulatory interactions of the enzyme with acidic phospholipids.

Amino Acid Sequence↗
PubMed1998

KQT2, a new putative potassium channel family produced by alternative splicing. Isolation, genomic structure, and alternative splicing of the putative potassium channels.

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of the testing membrane potential, and development of memory and learning. Eleven mouse cDNA clones homologous to the new human putative K+ channel (designated HNSPC, which we recently reported) were isolated from the brain cDNA libraries. All these proteins coded by the isolated cDNAs were identical from the N-terminal to the sixth transmembrane domain, but exhibited differences in the sequence and length of the C-terminal cytoplasmic region. Analyses of the mouse genomic DNAs showed that these clones originated from a single gene located on mouse chromosome 2H3-4, which proved that these clones were generated by alternative RNA splicing. Since all isoforms showed significant structural identity with KVLQT1 (64% identity in the transmembrane domains), which is known to associate with IsK, they were designated mKQT2.1-mKQT2.11. Northern blot analysis indicated that the mRNAs of the mKQT2 isoforms were exclusively expressed in the brain. In the mouse cerebellum region, the localized expression of these clones in the Purkinje cell layer and Golgi cells was shown by in situ hybridization analysis. These transcripts were also detected in the mouse embryonic developmental stage (11th, 15th and 17th day); and in particular, the mRNAs for shorter forms (mKQT2.9, mKQT2.10 or mKQT2.11) were abundantly found on the 11th day after gestation. Although these mKQT2 isoforms had the characteristic structure of voltage-gated K+ channels, functional expression of K+ currents were not detected in Xenopus oocytes.

Alternative Splicing↗