SEARCH · PubMed Health

Results for “Alternative Splicing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources

PubMed1999

[Cloning of full-length Dnmt3b cDNA and its alternative splicing isoforms in mouse embryo].

OBJECTIVE: To clone the novel full-length cDNA related to mouse embryonic development. METHODS: Starting from a new expression sequence (clone 2 #) out of a mouse embryonic cDNA library, its full-length cDNA were obtained by rapid amplification of cDNA ends with polyA RNA isolated from mouse 8-9 day embryonic tissue as template, combined with screening of mouse embryonic cDNA library by PCR. Based on the sequences at the two ends of the cDNA, the primers for long range RT-PCR were designed to confirm its factuality. RESULTS: The cDNA isolated in our hands was one of the alternative splicing isoforms of mouse de novo DNA cytosine 5'-specific methyltransferase gene (Dnmt3b) reported in July, 1998. A new alternative splicing region upstream of its initiate codon was found 5' forwards 152 bp over the Dnmt3b cDNA sequence. Eight alternative splicing isoforms of Dnmt3b from mouse embryonic tissue were cloned (Genbank accession number are AF151969, AF151970, AF151971, AF151972, AF151973, AF151974, AF151975, AF151976 respectively). CONCLUSIONS: The 4 kb full-length Dnmt3b cDNA was isolated. Dnmt3b may have novel exons at its 5' upstream and present a complex alternative splicing pattern during mouse embryonic development.

Alternative Splicing↗

PubMedDate not supplied

[Different patterns of evolution in alternative and constitutive coding regions of Drosophila alternatively spliced genes].

Seven hundred and ninety Drosophila melanogaster genes, alternatively spliced in coding regions were considered together with their Drosophila pseudoobscura orthologs. It was found that nucleotide substitutions in alternative coding regions accumulate more intensively than in constitutive regions. Moreover, the evolutionary pattern of alternative regions depends on their inclusion mechanisms (use of alternative promoters, splicing sites or polyadenylation sites) significantly. The rate of synonymous substitutions varies is more dramatically than that of nonsynonymous substitutions. Nucleotide substitution patterns in different classes of alternative regions of mammalian and Drosophila genes have little in common.

Alternative Splicing↗

PubMed1999

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Alternative Splicing↗

PubMed1994

Alternative splicing of the guanine nucleotide-binding regulatory protein Go alpha generates four distinct mRNAs.

Go alpha a guanine nucleotide-binding (G) protein abundant in brain and other neural tissues, has been implicated in ion channel regulation. Concerted efforts in several laboratories have revealed multiple Go alpha mRNAs and protein isoforms in different contexts. Go alpha is a single copy gene in mammalian species, although the structure, number and tissue localization of Go alpha mRNAs reported by investigators are inconsistent. To define the cell-specific expression of alternatively spliced variants of Go alpha mRNA, we employed several strategies, including Northern hybridizations with sequences-specific oligonucleotides, selective digestions of Go alpha mRNA using RNase H, and adaptations of the polymerase chain reaction. Four distinct alternatively spliced variants were identified, a 5.7-kb Go alpha 2 mRNA and three Go alpha 1 mRNAs with different 3' UTRs. The UTRs of the three Go alpha 1s are composed of different combinations of what have been referred to as UTR-A and UTR-B. The sequences of the spliced segments are well conserved among mammalian species, suggesting a functional role for these alternatively spliced 3' UTRs in post-transcriptional and/or tissue-specific regulation of Go alpha expression. The position of the intron-exon splice boundary at nucleotide 31 following T of the TGA stop codon is conserved in the Gi alpha 2 and Gi alpha 3 genes, consistent with the notion that similar alternative splicing of 3' UTRs occurs in products of these related genes.

Alternative Splicing↗

PubMed2001

Alternative splicing switches potassium channel sensitivity to protein phosphorylation.

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.

Alternative Splicing↗

PubMed2006

Generation of interleukin-17 receptor-like protein (IL-17RL) in prostate by alternative splicing of RNA.

BACKGROUND: Interleukin-17 receptor-like protein (IL-17RL) expressed in prostate tissues changes with advanced cancers due to extensive alternative splicing, which affects the final protein. Predominant IL-17RL splice isoform variants have not been identified, hindering functional studies. METHODS: A cDNA library of IL-17RL transcripts was arrayed onto nylon membranes. Individual transcript exon structures were determined by successively probing membranes with exon-specific oligonucleotides. The most common variants were transiently over-expressed in 293T cells. RESULTS: We detected >90 different IL-17RL isoforms. Three most abundant isoforms account for approximately half the total transcripts; the full-length variant just over 11%. Surprisingly, most alternative splicing does not alter the reading frame of the full-length molecule; therefore, resulting proteins vary mostly in N-terminal domains. CONCLUSIONS: IL-17RL exists as multiple isoforms due to extensive alternative splicing. We identified the most abundant splices in prostate tissue and established a technique to investigate changes in RNA IL-17RL splicing that occur in advanced cancers.

Alternative Splicing↗

PubMed2000

Alternative splicing of the first F3 domain from chicken collagen XIV affects cell adhesion and heparin binding.

The N terminus of chicken collagen XIV is subject to alternative splicing. The longer isoform contains a fibronectin type III (F3) domain at its N terminus, whereas the shorter isoform is lacking this domain. Alternative splicing of the F3 domain is developmentally regulated. At early embryonic stages, both isoforms are expressed, whereas after hatching only the longer isoform is expressed. When immobilized on plastic dishes, the recombinant F3 domain promotes the adhesion of mesenchymal cells. Attachment to this domain is specifically inhibited by heparin but not by other glycosaminoglycans. Molecular modeling studies illustrate that the first F3 domain harbors a positively charged groove, which may accommodate the negatively charged heparin chain. Site-directed mutagenesis of a single lysine residue within this groove abolishes the cell binding activity but does not affect the heparin binding activity. Cell binding and heparin binding are therefore two functionally distinct properties shared by the N-terminal F3 domain. When full-length collagen XIV polypeptides that either contain or lack the first F3 domain are tested on heparin-Sepharose, a pronounced difference in their relative affinity is observed. Thus, alternative splicing of the N-terminal F3 domain influences the interaction of this FACIT (fibril-associated collagens with interrupted triple helices) collagen with cells and with glycosaminoglycans.

Alternative Splicing↗

PubMed2004

Alternative splicing of the multidrug resistance protein 1/ATP binding cassette transporter subfamily gene in ovarian cancer creates functional splice variants and is associated with increased expression of the splicing factors PTB and SRp20.

PURPOSE: Overexpression of multidrug resistance protein 1 (MRP1) confers resistance to a range of chemotherapeutic agents in cell lines and could be involved in clinical drug resistance of some tumor types also. We examined MRP1 expression in a small series of untreated human ovarian tumors and matched normal tissues. EXPERIMENTAL DESIGN: We analyzed ten pairs of snap-frozen ovarian tumor and matched normal total ovarian tissues from the same patients for expression of MRP1 by reverse transcription-PCR. Amplified PCR products were sequenced to reveal splicing events of MRP1. MRP1 splice variants were expressed as enhanced green fluorescent fusion proteins in HEK293T cells to demonstrate their localization in the cell and their activity in conferring resistance to doxorubicin. The expression of splicing factors PTB and SRp20 was examined by Western blot. RESULTS: MRP1 was expressed in all 10 of the pairs of specimens. Multiple MRP1 cDNA fragments of various sizes were amplified between exons 10 and 19. Of interest, more MRP1 cDNA fragments were detected in ovarian tumors than in matched normal tissues in 9 of 10 pairs. We identified 10 splicing forms between exons 10 and 19 of the MRP1 gene with exon skipping ranging from 1 to 7. Amplification of the entire coding region of MRP1 from 1 ovarian tumor revealed >20 splice variants. We found whole and partial exon skipping and partial intron inclusion in these splice variants. We expressed 3 of these MRP1 splice variants in HEK293T cells and found that they appeared to localize to the plasma membrane and were functional in conferring resistance to doxorubicin. In addition, we identified a few nucleotide variations in this gene. To understand the basis for increased splice variants in the tumors, we examined splicing factor expression in these tissues. Western blot analysis revealed that two splicing factors, PTB and SRp20, were overexpressed in most ovarian tumors compared with their matched normal ovarian tissues. Importantly, overexpression of both of these splicing factors was associated with the increased number of MRP1 splicing forms in the ovarian tissues. CONCLUSION: The MRP1 gene undergoes alternative splicing at a higher frequency in ovarian tumors than in matched normal tissues. Some of these splice variants confer resistance to doxorubicin. Expression of splicing factors PTB and SRp20 is strongly associated with the alternative splicing of the MRP1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties.

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na(+)-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the approximately 19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Alternative Splicing↗

PubMed2006

Genotoxic stress induces coordinately regulated alternative splicing of the p53 modulators MDM2 and MDM4.

The tumor suppressor protein p53 is a transcription factor that induces G(1) arrest of the cell cycle and/or apoptosis. The murine double-minute protein MDM2 and its homologue MDM4 (also known as MDMX) are critical regulators of p53. Altered transcripts of the human homologue of mdm2, MDM2, have been identified in human tumors, such as invasive carcinoma of the breast, lung carcinoma, and liposarcoma. MDM2 alternate forms act to negatively regulate the normal MDM2 gene product, thus activating p53. Although many reports have documented a plethora of tumor types characterized by MDM2 alternative transcripts, few have investigated the signals that might initiate alternative splicing. We have identified a novel role of these alternative MDM2 transcripts in the normal surveillance mechanism of the cell and in DNA damage response. We report that alternate forms of MDM2 are detected after UV irradiation. Furthermore, we show that mouse cells treated with UV are also characterized by alternative transcripts of mdm2, suggesting that this is an important and evolutionarily conserved mechanism for regulating the expression of MDM2/mdm2. An additional p53 regulator and mdm2 family member, MDM4, is likewise alternatively spliced following UV irradiation. By activating alternative splicing of both MDM2 and MDM4, yet another layer of p53 regulation is initiated by the cells in response to damage. A stepwise model for malignant conversion by which alternate forms of MDM2 and MDM4 place selective pressure on the cells to acquire additional alterations in the p53 pathway is herein proposed.

Alternative Splicing↗

PubMed2006

Alternative splicing increases complexity of stem cell transcriptome.

Development of highly anticipated stem cell-based therapies requires a detailed understanding of mechanisms regulating biological properties of these cells. Comprehensive identification of all biological molecules produced in stem cells is an important step toward this goal. During the past several years, microarray studies have essentially identified genes that are transcriptionally activated in various embryonic and adult stem cell populations. However, the extent of post-transcriptional modifications within the stem cell transcriptome remained undetermined. Recently, we presented evidence that thousands of genes expressed in hematopoietic and embryonic stem cells undergo alternative splicing. Using combined computational and experimental analyses, we found that the frequency of alternative splicing is especially high in tissue-specific genes, as compared to ubiquitous genes. Our results also indicate that negative regulation of constitutively active splicing sites can be a prevalent mode for generation of splicing variants, and that alternative splicing is generally not conserved between orthologous genes in human and mouse. Here, we discuss the implications of our findings for stem cell biology, and present possible approaches toward genome-wide identification and characterization of splice variants.

Alternative Splicing↗

PubMed2005

Increased expression and activity of CDC25C phosphatase and an alternatively spliced variant in prostate cancer.

Alterations in the control of cell cycle progression have been implicated in a wide variety of malignant neoplasms, including prostate cancer. CDC25 phosphatases belong to the tyrosine phosphatase family and play a critical role in regulating cell cycle progression by dephosphorylating cyclin-dependent kinases at inhibitory residues. CDC25C plays an important role in the G2-M transition by activating Cdc2/Cyclin B1 complexes. To determine whether CDC25C activity is altered in prostate cancer, we have examined the expression of CDC25C and an alternatively spliced variant in human prostate cancer samples and cell lines. CDC25C protein is up-regulated in prostate cancer in comparison with normal prostate tissue and is present almost exclusively in its active dephosphorylated form. Expression of a biologically active alternatively spliced CDC25C isoform is also increased in prostate cancer and expression of alternatively spliced CDC25C is correlated to occurrence of biochemical (prostate-specific antigen) recurrence. We have also developed a quantitative reverse transcriptase-PCR analysis of Ki-67 expression as a method of measuring proliferative activity in prostate cancer from RNA samples. Based on this analysis of Ki67 expression, some but not all of this increase in CDC25C and its alternatively spliced variants is correlated with increased proliferation in prostate cancer. This data suggests that CDC25C might play an important role in prostate cancer progression and could be used to monitor and predict the aggressiveness of this disease.

Alternative Splicing↗

PubMed1996

[Higher expression levels of alternatively spliced pX mRNA in human T lymphotropic virus type I (HTLV-I) asymptomatic carriers positive for antibodies to p40tax protein].

We cloned and sequenced complementary DNA (cDNA) of human T lymphotropic virus type I (HTLV-I) pX gene mRNA expressed in peripheral blood lymphocytes of asymptomatic carriers and searched for a possible correlation between HTLV-I pX mRNA expression levels to viral load and anti-p40tax status. Nine cDNA clones had four alternatively spliced forms of mRNA, three forms were identical to the reported cDNA sequences. One cDNA clone contained a novel splicing acceptor site, thereby indicating an unidentified form of pX mRNA, termed as pX delta 17 delta 37. These results, together with previous observations, suggest that HTLV-I has complex pattern of splicing. The expression levels of the five pX mRNAs, pX, pX delta 17, p21rex, orf II, and pX delta 17 delta 37, were examined in twenty-one asymptomatic carriers. Alternatively spliced pX mRNA species were detected in all the carriers. Semiquantitation of pX mRNAs revealed that pX and pX delta 17 were the dominant mRNA species among alternatively spliced pX mRNA. All pX mRNAs but the orf II message correlated with amounts of proviral DNA significantly (p < 0.05). The levels of proviral DNA and mRNAs were significantly higher in anti-p40tax positive carriers than in negative ones. These observations suggest that the alternatively spliced pX mRNAs are expressed ubiquitously in the peripheral blood lymphocytes of HTLV-I asymptomatic carriers with complex pattern of splicing and that the presence of anti-p40tax may be a marker for a higher viral load and viral replication levels in HTLV-I asymptomatic carriers. Our observations may explain previous finding that the carriers rate is higher in the offspring of anti-p40tax positive mothers than those of anti-p40tax negative mothers.

Aged↗

PubMed2004

Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.

Alternative splicing of the P/Q-type channel (Ca(V)2.1) promises customization of the computational repertoire of neurons. Here we report that concerted splicing of its main alpha1A subunit, at both an EF-hand-like domain and the channel C terminus, controls the form of Ca2+-dependent facilitation (CDF), an activity-dependent enhancement of channel opening that is triggered by calmodulin. In recombinant channels, such alternative splicing switches CDF among three modes: (1) completely "ON" and driven by local Ca2+ influx through individual channels, (2) completely "OFF," and (3) partially OFF but inducible by elevated global Ca2+ influx. Conversion from modes 1 to 3 represents an unprecedented dimension of control. The physiological function of these variants is likely important, because we find that the distribution of EF-hand splice variants is strikingly heterogeneous in the human brain, varying both across regions and during development.

Alternative Splicing↗

PubMed1988

The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing.

The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

Animals↗

PubMed2002

Alternative splicing of type II procollagen gene in the dedifferentiation of rat epiphyseal chondrocytes serially cultured in monolayer.

Type II procollagen is synthesized in two forms (IIA and IIB), generated by the alternative splicing of its precursor mRNA. The purpose of this study was to determine whether alternative splicing of type II procollagen pre-mRNA occurs during the dedifferentiation of chondrocytes in monolayer culture. We serially cultured rat epiphyseal chondrocytes for eight weeks in monolayer, and investigated the expression patterns of the two forms of type II procollagen transcripts according to the duration of the culture period. Reverse transcription-polymerase chain reaction and northern analysis revealed that both transcripts peaked on the day of cell isolation, but the expression level of IIB form was much higher than that of IIA form. The expression of IIA form decreased gradually, whereas the expression of IIB form decreased so rapidly that the ratio of type IIB and type IIA transcripts was reversed in the second week. In situ hybridization showed that all cells expressed type IIA mRNA throughout the culture period. The signal intensity for type IIA transcript decreased gradually in accordance with the results of RT-PCR and northern analysis. In situ hybridization of type IIB mRNA showed different results from in situ hybridization of type IIA mRNA. Like type IIA transcript, the freshly isolated chondrocytes and the cells in the first week showed positive signals for type IIB transcript. However, in the second week, positive signal was detected neither in polygonal cells nor large fibroblastic cells. Only a few cells of small size were weakly positive. From the third to the eighth week, no positive signal was detected. These findings indicate that alternative splicing of type II procollagen gene does occur and is regulated during the dedifferentiation of chondrocytes in monolayer culture.

Alternative Splicing↗

PubMed1991

An alternative spliced form of FosB is a negative regulator of transcriptional activation and transformation by Fos proteins.

Two forms of FosB transcript and their products can be identified in mouse NIH 3T3 cells following serum induction. The larger RNA codes for a 338-amino acid protein, whereas the smaller RNA results from the removal of an additional 140 nucleotides from FosB mRNA by alternative splicing. This alternative splicing event places a stop codon following the "leucine zipper" region and results in a shorter protein (FosB2) of 237 amino acids that lacks 101 amino acids at the carboxyl terminus. FosB2 is able to form heterodimers with c-Jun and bind to an AP-1 site but is not able to activate the transcription of promoters containing AP-1 sites. Furthermore, FosB2 can not only suppress the transcriptional activation by c-Fos and c-Jun of promoters containing an AP-1 site but also interferes with the transforming potential of viral and cellular Fos proteins. We propose that FosB2 protein functions as a trans-negative regulator.

Amino Acid Sequence↗

PubMed1993

Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect.

Approximately 2% of Caucasians and African-Americans are homozygous for a nonsense mutation in exon 2 of the AMPD1 (AMP deaminase) gene. These individuals have a high grade deficiency of AMPD activity in their skeletal muscle. More than 100 patients with AMPD1 deficiency have been reported to have symptoms of a metabolic myopathy, but it is apparent many individuals with this inherited defect are asymptomatic given the prevalence of this mutant. Results of the present study provide a potential molecular explanation for "correction" of this genetic defect. Alternative splicing eliminates exon 2 in 0.6-2% of AMPD1 mRNA transcripts in adult skeletal muscle. Expression studies document that AMPD1 mRNA, which has exon 2 deleted, encodes a functional AMPD peptide. A much higher percentage of alternatively spliced transcripts are found during differentiation of human myocytes in vitro. Transfection studies with human minigene constructs demonstrate that alternative splicing of the primary transcript of human AMPD1 is controlled by tissue-specific and stage-specific signals. Alternative splicing of exon 2 in individuals who have inherited this defect provides a mechanism for phenotypic rescue and variations in splicing patterns may contribute to the variability in clinical symptoms.

AMP Deaminase↗