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 757 records · Page 42Linked to original sources

PubMed1993

Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript.

Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library. Both clones are incomplete and appear to result from alternative splicing. One clone is missing the sequence encoding exon 4 and the other is missing exon 8. The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones. This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process. By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli. In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.

Alternative Splicing↗

PubMed1988

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.

Animals↗

PubMed2005

Mouse p10, an alternative spliced form of p15INK4b, inhibits cell cycle progression and malignant transformation.

The INK4 family of proteins negatively regulates cell cycle progression at the G(1)-S transition by inhibiting cyclin-dependent kinases. Two of these cell cycle inhibitors, p16(INK4A) and p15(INK4B), have tumor suppressor activities and are inactivated in human cancer. Interestingly, both INK4 genes express alternative splicing variants. In addition to p16(INK4A), the INK4A locus encodes a splice variant, termed p12--specifically expressed in human pancreas--and ARF, a protein encoded by an alternative reading frame that acts as a tumor suppressor through the p53 pathway. Similarly, the human INK4B locus encodes the p15(INK4B) tumor suppressor and one alternatively spliced form, termed as p10. We show here that p10, which arises from the use of an alternative splice donor site within intron 1, is conserved in the mouse genome and is widely expressed in mouse tissues. Similarly to mouse p15(INK4B), p10 expression is also induced by oncogenic insults and transforming growth factor-beta treatment and acts as a cell cycle inhibitor. Importantly, we show that mouse p10 is able to induce cell cycle arrest in a p53-dependent manner. We also show that mouse p10 is able to inhibit foci formation and anchorage-independent growth in wild-type mouse embryonic fibroblasts, and that these antitransforming properties of mouse p10 are also p53-dependent. These results indicate that the INK4B locus, similarly to INK4A-ARF, harbors two different splicing variants that can be involved in the regulation of both the p53 and retinoblastoma pathways, the two major molecular pathways in tumor suppression.

Alternative Splicing↗

PubMed2005

Promoter usage and alternative splicing.

Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

Alternative Splicing↗

PubMed2002

Alternative splicing isoform of tau protein kinase I/glycogen synthase kinase 3beta.

Glycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer-like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI)/GSK3beta with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences. The variant protein, named TPKI2/GSK3beta2, was abundant in the brain. Immunohistochemistry indicated differential distribution of the conventional and the new TPKI/GSK3beta isoforms within young neurons. TPKI2/GSK3beta2 showed decreased kinase activities towards two phosphorylation sites on tau compared with the conventional isoform. Immunohistochemistry indicated that TPKI2/GSK3beta2 occurs predominantly in the neuronal soma, while TPKI1/GSK3beta1 is found both in the soma and processes. These results indicate that the new splice isoform has a different function. Because the amino acid insert occurs in the domain implicated in interaction with a protein phosphatase in a homologous kinase cdk-2, the alternative splicing can regulate multiprotein complex formation and function involving TPKI/GSK3beta.

Alternative Splicing↗

PubMed1992

Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity.

A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene--multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5' end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180, 156, 141, 74, and 59 amino acids in length.

Alternative Splicing↗

PubMed1997

CD44 isoform expression in periodontal tissues: cell-type specific regulation of alternative splicing.

CD44 functions as a receptor for various extracellular matrices and plays crucial roles in homotypic and heterotypic cell-cell interactions. Recently, the molecular structure of CD44 has been extensively analyzed and multiple isoforms produced by alternative splicing of messenger RNA have been identified. In this study, we examined the expression of CD44 isoforms on different cell types isolated from periodontal tissue. In order to examine tissue differences in CD44 isoform expression, we established in vitro cell culture of human gingival fibroblasts (HGF), human periodontal ligament cells (HPDL) and human gingival epithelial cells (HGEC). These cells all expressed CD44 protein and messenger RNA. However, immunoprecipitation and Northern blot analysis revealed that HGEC expressed larger CD44 isoforms than HGF and HPDL. Reverse transcription-polymerase chain reaction with primers flanking the insertion site of alternatively spliced exons was used to study details of the heterogeneity. All cells examined expressed a major band in the absence of alternatively spliced exons and additional larger bands. In particular, HGEC contained more abundant high molecular mass species. In vitro stimulation by IL-1 beta, TNF alpha or phorbol 12-myristate 13-acetate induced an increase in total CD44 messenger RNA in HGF but not change in overall patterns of CD44 isoform expression. However, the isoform expression of HGEC was sensitive to cell density. The amount of larger isoform was decreased by culturing cells beyond confluence. These findings suggest that CD44 isoform expression is cell type-specifically regulated in periodontium and altered according to growth phase of HGEC.

Alternative Splicing↗

PubMed2001

The distribution and function of alternatively spliced insertions in hDlg.

hDlg is the human homolog of the Drosophila Discs-large tumor suppressor. As a member of the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins, hDlg is composed of three PDZ (PSD-95, Dlg, and ZO-1) repeats, an SH3 (Src homology 3) motif, and a GUK (guanylate kinase-like) domain. Additionally, hDlg contains two regions of alternative splicing. Here we identify a novel insertion, I1B, located N-terminal to the PDZ repeats. We further analyze the tissue-specific combinations of insertions and correlate those results with the distribution of protein isoforms. We also identify the functions of the two alternatively spliced regions. The N-terminal alternatively spliced region is capable of binding several SH3 domains and also moderates the level of protein oligomerization. Insertions in the second region are responsible for determining the localization of hDlg, with insertion I3 targeting the protein to the membrane regions of cell-cell contact and insertion I2 targeting the protein to the nucleus.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis.

Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, mIL-1RAcP mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.

Alternative Splicing↗

PubMed2001

Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.

Alternative Splicing↗

PubMed2006

A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues.

BACKGROUND: Mammalian Gli proteins are important transcription factors involved in the regulation of Sonic hedgehog signal transduction pathway. Association of Gli2 with mammalian development and human disease led us to study the structure and expression of the human GLI2. RESULTS: We show that the region encoding GLI2 repressor domain is subject to alternative splicing in the gonadal tissues and different cell lines. Two major alternatively spliced forms of GLI2 mRNA arise from skipping exon 3 (GLI2Delta3) or exons 4 and 5 (GLI2Delta4-5). Both forms contain premature translational stop codons in the GLI2 open reading frame (ORF) starting from exon 2. Translation of GLI2Delta3 and GLI2Delta4-5 in vitro, initiated from downstream AUG codons, produced N-terminally truncated proteins. In Gli-dependent transactivation assay, expression of GLI2Delta3 induced activation of the reporter gene similar to that of the full-length construct (GLI2fl) containing complete ORF. However, expression of the GLI2Delta4-5 resulted in about 10-fold increase in activation, suggesting that deletion of the major part of repressor domain was responsible for the enhanced activation of GLI2 protein. CONCLUSION: Our data suggest that in addition to proteolytic processing, alternative splicing may be another important regulatory mechanism for the modulation of repressor and activator properties of GLI2 protein.

3' Untranslated Regions↗

PubMed2002

Alternative splicing of the beta 4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating.

Ca2+ channel beta subunits are important molecular determinants of the kinetics and voltage dependence of Ca2+ channel gating. Through direct interactions with channel-forming alpha1 subunits, beta subunits enhance expression levels, accelerate activation, and have variable effects on inactivation. Four distinct beta subunit genes each encode five homologous sequence domains (D1-5), three of which (D1, D3, and D5) undergo alternative splicing. We have isolated from human spinal cord a novel alternatively spliced beta4 subunit containing a short form of domain D1 (beta4a) that is highly homologous to N termini of Xenopus and rat beta3 subunits. The purpose of this study was to compare the gating properties of various alpha1 subunit complexes containing beta4a with those of complexes containing a beta4 subunit with a longer form of domain D1, beta4b. Expression in Xenopus oocytes revealed that, relative to alpha1A and alpha1B complexes containing beta4b, the voltage dependence of activation and inactivation of complexes containing beta4a were shifted to more depolarized potentials. Moreover, alpha1A and alpha1B complexes containing beta4a inactivated at a faster rate. Interestingly, beta4 subunit alternative splicing did not influence the gating properties of alpha1C and alpha1E subunits. Experiments with beta4 deletion mutants revealed that both the N and C termini of the beta4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are alpha1 subunit specific. Our data are best explained by a model in which distinct modes of activation and inactivation result from beta-subunit splice variant-specific interactions with an alpha1 subunit gating structure.

Adolescent↗

PubMed2005

Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products.

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

Alternative Splicing↗

PubMed2000

The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing.

We recently characterized a novel protein, GIT1, that interacts with G protein-coupled receptor kinases and possesses ADP-ribosylation factor (ARF) GTPase-activating protein activity. A second ubiquitously expressed member of the GIT protein family, GIT2, has been identified in data base searches. GIT2 undergoes extensive alternative splicing and exists in at least 10 and potentially as many as 33 distinct forms. The longest form of GIT2 is colinear with GIT1 and shares the same domain structure, whereas one major splice variant prominent in immune tissues completely lacks the carboxyl-terminal domain. The other 32 potential variants arise from the independent alternative splicing of five internal regions in the center of the molecule but share both the amino-terminal ARF GTPase-activating protein domain and carboxyl-terminal domain. Both the long and short carboxyl-terminal variants of GIT2 are active as GTPase-activating proteins for ARF1, and both also interact with G protein-coupled receptor kinase 2 and with p21-activated kinase-interacting exchange factors complexed with p21-activated kinase but not with paxillin. Cellular overexpression of the longest variant of GIT2 leads to inhibition of beta(2)-adrenergic receptor sequestration, whereas the shortest splice variant appears inactive. Although GIT2 shares many properties with GIT1, it also exhibits both structural and functional diversity due to tissue-specific alternative splicing.

ADP-Ribosylation Factors↗

PubMed1999

Analysis of the kinase-related protein gene found at human chromosome 3q21 in a multi-gene cluster: organization, expression, alternative splicing, and polymorphic marker.

We report the amino acid sequence, genomic organization, tissue expression, and alternative splice patterns for the human kinase related protein (KRP) gene, as well as the discovery of a new CA repeat sequence polymorphic marker in an upstream intron of the myosin light chain kinase (MLCK) gene. The KRP/MLCK genetic locus is a prototype for a recently discovered paradigm in which an independently regulated gene for a non-enzymic protein is embedded within a larger gene for a signal transduction enzyme, and both classes of proteins are involved in the regulation of the same cellular structure. The MLCK/KRP gene cluster has been found only in higher vertebrates and is localized to human chromosome 3q21. The determination of the human KRP amino acid sequence through cDNA sequence analysis and its comparison to the exon/intron organization of the human KRP gene revealed an alternative splice pattern at the start of KRP exon 2, resulting in the insertion of a single glutamic acid in the middle of the protein. Examination of tissue distribution using Northern blot analysis revealed that the human expression pattern is more similar to the well-characterized chicken KRP gene expression pattern than to rodent or rabbit. Unexpected differences of the human gene from other species is the apparent expression of the human gene products in adult cardiac muscle, an observation that was pursued further by the production of a site-directed antiserum and immunohistochemistry analysis. The results reported here provide insight into the conserved and variable features of this late evolving genetic paradigm, raise new questions about the molecular aspects of cardiac muscle regulation, and provide tools needed for future clinical studies. The comparative analysis of the MLCK/KRP locus, combined with the recent discovery of a similar genomic relationship among other signal transduction proteins, suggest a diverse distribution of this theme among signal transduction systems in higher vertebrate genomes and indicate the utility of comparative genomics in revealing late evolving genetic paradigms.

Adult↗

PubMed1994

Modulation of human saposin B sphingolipid-binding specificity by alternative splicing. A study with saposin B-derived synthetic peptides.

The saposins A, B, C, and D, produced by proteolytic maturation of the same precursor protein, prosaposin, are sphingolipid-binding proteins which function as activators for lysosomal enzymes involved in sphingolipid hydrolysis. The alternative splicing of the prosaposin gene results in the inclusion or exclusion of exon 8 into transcribed prosaposin mRNA through the use of alternative acceptor sites. The relative abundance of each alternatively spliced mRNA was determined by reverse transcription-polymerase chain reaction in various human tissues and cell lines. Exon 8 codes for only three amino acid residues, Gln-Asp-Gln, in the saposin B domain of prosaposin. The prosaposin mRNA containing exon 8 is the major species in cultured skin fibroblasts, brain, and pituitary glands together with a smaller amount of mRNA devoid of exon 8, whereas the prosaposin mRNA detected in liver and lymphoblasts was devoid of exon 8 insertion. Previous structural modeling studies on saposin B have suggested that the Gln-Asp-Gln insertion occurs in an amphipathic alpha-helix region of the protein which is implicated in the binding of GM1-ganglioside. We report that synthetic peptides containing the alpha-helix, with and without the Gln-Asp-Gln insertion, have different binding affinities for GM1-ganglioside, sulfatide, and sphingomyelin. The insertion of the Gln-Asp-Gln sequence completely abolishes the capacity of the peptide to bind GM1-ganglioside, whereas its affinity for sulfatide and sphingomyelin is increased about 4-fold and almost 2-fold, respectively. No significant binding of glucosylceramide was observed with both peptides. These results suggest that alternative splicing of prosaposin mRNA may change binding specificity of saposin B presumably to adapt to the variable sphingolipid composition of tissues.

Alternative Splicing↗

PubMed1998

The human noradrenaline transporter gene contains multiple polyadenylation sites and two alternatively spliced C-terminal exons.

Sequencing downstream of the C-terminal exon 14 of the human noradrenaline transporter (hNAT) gene reveals 5 consensus polyadenylation signals, several adenylate/uridylate-rich elements (AREs) and a new C-terminal exon, designated as exon 15. The tandemly arranged polyadenylation sites are in good conformity with the 3.6- and 5.8-kb hNAT mRNA transcripts. Expression of the alternatively spliced C-terminal exon 15 is shown by RT-PCR. This alternative splicing event proposes additional hNAT mRNA species of 2.4-3 kb in size. Corresponding NAT transcripts are found by Northern analysis of human SKN and rat PC12 cell RNA. Sequence comparison of the hNAT gene to two bovine NAT cDNAs shows the interspecies conservation of this alternative splicing event, the close relationship of human and bovine NAT genes, and implicates a functional role for the transporters C-terminal domain.

Alternative Splicing↗

PubMed1998

The human DENN gene: genomic organization, alternative splicing, and localization to chromosome 11p11.21-p11.22.

We have previously isolated and sequenced the cDNA of a novel gene, DENN, that exhibits differential mRNA expression in normal and neoplastic cells. The open reading frame of 4761 nucleotides encodes a putative hydrophilic protein of 1587 amino acids with a calculated molecular mass of 176,431 Da. Within DENN cDNA lies an alternative exon segment of 129 nucleotides encoding 43 amino acids, which may be excluded from some transcripts by alternative splicing. The serine- and leucine-rich DENN protein possesses a RGD cellular adhesion motif and a leucine-zipper-like motif associated with protein dimerization, and shows partial homology to the receptor binding domain of tumor necrosis factor alpha. DENN is virtually identical to MADD, a human MAP kinase-activating death domain protein that interacts with type I tumor necrosis factor receptor. DENN displays significant homology to Rab3 GEP, a rat GDP/GTP exchange protein specific for Rab3 small G proteins implicated in intracellular vesicle trafficking. DENN also exhibits strong similarity to Caenorhabditis elegans AEX-3, which interacts with Rab3 to regulate synaptic vesicle release. Composed of 15 exons (ranging in size from 73 to 1230 bp) and 14 introns (varying from about 170 bp to 5.3 kb), the DENN gene is estimated to span at least 28 kb. The alternative splicing event was traced to an alternative 5' donor site involving exon 7. DENN was mapped to chromosome region 11p11.21-p11.22 by FISH. Using polyclonal antibodies against a synthetic peptide, Western blotting of MOLT-4 T-lymphoblastic leukemic cell proteins and immunoblotting of subcellular fractions of MOLT-4 cells and PLC/PRF/5 liver cancer cells yielded data corroborating the alternative splicing mechanism that generates two variant isoforms of the DENN protein that display differential expression in cells of different lineages.

Alternative Splicing↗