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Results for “Alternative Splicing”

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PubMed2002

Alternative splicing and interaction with di- and polyvalent cations control the dynamic range of acid-sensing ion channel 1 (ASIC1).

Homomeric acid-sensing ion channel 1 (ASIC1) can be activated by extracellular H(+) in the physiological pH range and may, therefore, contribute to neurotransmission and peripheral pain perception. ASIC1a and ASIC1b are alternative splice products of the ASIC1 gene. Here we show that both splice variants show steady-state inactivation when exposed to slightly decreased pH, limiting their operational range. Compared with ASIC1a, steady-state inactivation and pH activation of ASIC1b are shifted to more acidic values by 0.25 and 0.7 pH units, respectively, extending the dynamic range of ASIC1. Shifts of inactivation and activation are intimately linked; only two amino acids in the ectodomain, which are exchanged by alternative splicing, control both properties. Moreover, we show that extracellular, divalent cations like Ca(2+) and Mg(2+) as well as the polyvalent cation spermine shift the steady-state inactivation of ASIC1a and ASIC1b to more acidic values. This leads to a potentiation of the channel response and is due to a stabilization of the resting state. Our results indicate that ASIC1b is an effective sensor of transient H(+) signals during slight acidosis and that, in addition to alternative splicing, interaction with di- and polyvalent cations extends the dynamic range of ASIC H(+) sensors.

Acid Sensing Ion Channels↗

PubMed1997

Organization of the neurofascin gene and analysis of developmentally regulated alternative splicing.

Neurofascin is an axonal member of the L1 subgroup of the immunoglobulin superfamily implicated in neurite extension in the course of embryonic development. Here we have isolated and characterized the gene encoding chicken neurofascin. Comparison of genomic sequences with cDNA sequences provides the structure and localization of intron/exon boundaries and indicates that neurofascin isoforms are generated by alternative splicing of its pre-mRNA. The neurofascin gene is composed of 33 exons distributed over 72 kilobases. Each of the six immunoglobulin- and five fibronectin-type III-like domains is encoded by two exons. While introns between domains are of phase 1, others are of phase 0, 1, or 2. Alternative splicing of neurofascin is developmentally regulated as shown by polymerase chain reaction analysis. Furthermore, plasmid libraries from long range polymerase chain reaction-amplified cDNA of neurofascin were used to examine and quantify the distribution of alternatively spliced exons in individual neurofascin molecules. We found 50 different neurofascin isoforms at different developmental stages and revealed the existence of one major "early" in comparison with multiple "late" neurofascin isoforms.

Alternative Splicing↗

PubMed1994

Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms.

Minimal ectopic expression of the p58GTA protein kinase results in a provocative phenotype involving cell cycle delay, mitotic catastrophe, and decreased cell viability. In addition, this kinase is well conserved evolutionarily, ubiquitously expressed, and its genes map to a position on human chromosome 1 frequently deleted in the late stages of tumorigenesis. Here we report that the p58GTA protein kinase is a member of a larger subfamily of proteins. The mRNAs encoding these proteins are generated by alternative splicing from multiple duplicated genes. These isoforms range in size from 50 to 110 kDa. Divergence between the alternatively spliced isoforms is localized to the amino-terminal region of the molecule. The entire p58GTA open reading frame is conserved in most of these p58GTA isoforms. The predicted sequences of the larger isoforms encode bipartite nuclear localization signal sequences and extensive polyglutamic acid domains. Antibodies to the p58GTA isoform were used to confirm the presence of the alternatively spliced isoforms in different cell types as well as identify two additional isoforms that appear to arise from a separate gene(s). Cellular fractionation studies indicate that one of the isoforms is found only in the nucleus, and the remainder are found in both the cytoplasm and the nucleus. Expression and localization of some p58GTA isoforms suggest that they may have specialized cellular functions. Because of the large number of isoforms generated from multiple genes we propose naming these kinases PITSLRE alpha 1, alpha 2-1, alpha 2-2, alpha 2-3 alpha 2-4, beta 1, beta 2-1, and beta 2-2 based on the conserved sequence of the PSTAIRE box unique to p34cdc2 kinases and the gene from which they are transcribed.

Alternative Splicing↗

PubMed2006

Characterization of the desensitization properties of five dopamine receptor subtypes and alternatively spliced variants of dopamine D2 and D4 receptors.

Proper regulation of brain dopaminergic activity is essential for maintaining normal mental functions. In this study, the regulatory properties of five different dopamine receptor subtypes and alternative splicing variants of dopamine D2 and D4 were examined. The stimulation of D1R, D2R, D5R but not D3R, D4R caused the robust translocation of beta-arrestin to the plasma membrane. When D1R or D3R were co-expressed with D2R, D1R significantly inhibited the sequestration of D2R, suggesting that the inhibitory effects of D1R on the D2R sequestration could explain the synergistic activity between two receptors. The sequestration of alternatively spliced isoforms of D2R was differently regulated by GRKs and beta-arrestins. Three alternative splicing variants of D4R produced a similar level of beta-arrestin translocation, and the studies with the deletion mutants of D4R within the third cytoplasmic loop revealed that the regions containing the SH3-binding domains are responsible for the beta-arrestin translocation.

Animals↗

PubMed2003

Alternative splicing as a mechanism for regulating 14-3-3 binding: interactions between hD53 (TPD52L1) and 14-3-3 proteins.

D52 (TPD52)-like proteins are coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma, which have been proposed to represent signalling intermediates and regulators of vesicle trafficking. D52-like gene transcripts are subject to alternative splicing, with sequences encoding a region termed insert 3 being affected in all three D52-like genes. We have now identified a 14-3-3 binding motif within one of two alternatively spliced exons encoding insert 3. As predicted from the distribution of 14-3-3 binding motifs in four hD52-like bait proteins tested, only a hD53 isoform encoding a 14-3-3 binding motif bound both 14-3-3beta and 14-3-3zeta preys in the yeast two-hybrid system. Since D53 proteins carrying 14-3-3 binding motifs are predicted to be widely expressed, polyclonal antisera were derived to specifically detect these isoforms. Using soluble protein extracts from breast carcinoma cell lines, pull-down assays replicated interactions between recombinant 14-3-3beta and 14-3-3zeta isoforms and exogenously expressed hD53, and co-immunoprecipitation analyses demonstrated interactions between endogenous 14-3-3 and both endogenously and exogenously-expressed hD53 protein. Co-expressed hD53 and 14-3-3 proteins were similarly demonstrated to co-localise within the cytoplasm of breast carcinoma cell lines. These results identify 14-3-3 proteins as partners for hD53, and alternative splicing as a mechanism for regulating 14-3-3 binding.

14-3-3 Proteins↗

PubMed1995

Alternatively spliced human type 1 angiotensin II receptor mRNAs are translated at different efficiencies and encode two receptor isoforms.

The peptide hormone angiotensin II (AngII) plays a principal role in regulating blood pressure and fluid homeostasis. Most of its known effects are mediated by a guanine nucleotide-regulatory protein (G protein)-coupled receptor pharmacologically defined as the type-1 AngII receptor or AT1. Characterization of cDNA and genomic clones shows that the human AT1 gene contains five exons and encodes two receptor isoforms as a result of alternative splicing. Exon 5 contains the previously characterized open reading frame for AT1, and exons 1 to 3 are alternatively spliced upstream of it to generate several mRNA species, while transcripts containing exon 4 are of minor abundance. In an in vitro translation system, the presence of exon 1 was found to be extremely inhibitory to translation, probably because it can form a stable secondary structure at the RNA level. The alternatively spliced second exon also had a strong inhibitory effect on translation, presumably because it contains a minicistron commencing with an ATG in an optimal context for translation initiation. Exon 2 was similarly inhibitory to protein production in transfected cells, but exon 1 was found to enhance protein synthesis in this system. Transcripts containing exon 3 and 5, which comprise up to one-third of AT1 mRNAs in all tissues examined, encode a receptor with an amino-terminal extension of 32-35 amino acids. These transcripts were translated into a larger receptor isoform in vitro and produced a functional receptor with normal ligand binding and signaling properties in transfected cells.

Alternative Splicing↗

PubMed2006

Multiple alternative splicing in gonads of chicken DMRT1.

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla, although the existence of two sexes is nearly universal in the animal kingdom. However, the evolutionarily conserved DMRT1/dsx/mab3 with a common zinc finger-like DNA-binding motif, DM domain, share both similar structure and function between phyla. Here we report that six transcripts of the chicken DMRT1 were generated in gonads by multiple alternative splicing. By cDNA cloning and genomic structure analysis, we found that there were nine exons of DMRT1, which were involved in alternatively splicing to generate the DMRT1 transcripts. Northern blotting and reverse transcription (RT) PCR analysis revealed that the expression of chicken DMRT1 was testis-specific in adults. Whole-mount in situ hybridizations and RT-PCR indicated that DMRT1 b was specially expressed in embryo gonads and higher in male than female gonads at stage 31. The female gonad had stronger DMRT1 c expression than the male one, whereas DMRT1 f was detectable only in the male gonad at stage 31 of the key time of sex gonadal differentiation. The differential expression of these transcripts during gonadal differentiation provides new insight into roles of alternative splicing of DMRT1 in governing sex differentiation of the chicken.

Alternative Splicing↗

PubMed1994

Alternatively spliced mRNAs encoding soluble isoforms of the erythropoietin receptor in murine cell lines and bone marrow.

32D Epo and 32D GM cells are subclones of the murine 32D cell line which are selectively dependent for proliferation and survival on erythropoietin (Epo) or granulocyte/macrophage colony-stimulating factor (GM-CSF), respectively. 32D GM cells were previously shown to express significant levels of the Epo receptor mRNA and protein which was retained intracellularly and did not appear on the cell surface. We have now analyzed the EpoR mRNA from the 32D GM line, using PCR followed by direct sequencing. Several alternatively spliced products were detected. In some molecules, intron 5 (I5) or part of I6 or both were retained. Retention of I5 results in a mRNA potentially encoding an almost complete extracellular domain, while retention of I6 gives rise to a mRNA encoding the complete extracellular and transmembrane domains. A different type of splicing results in the loss of exon 5 (E5), giving rise to a sequence encoding a truncated extracellular domain. These alternatively spliced sequences are differentially represented in 32D Epo versus 32D GM cells. All are additionally present in normal bone marrow cells. Apart from these alternatively spliced EpoR RNAs, no other abnormalities were detected in EpoR RNA from 32D GM cells that could account for the intracellular retention of EpoR in the non-erythroid subclones of 32D.

Alternative Splicing↗

PubMed1992

Cis regulating elements which control in vivo alternative splicing of the chicken beta tropomyosin primary transcript.

The beta tropomyosin gene of the chicken contains a pair of alternatively spliced mutually exclusive exons the use of which is developmentally regulated. Exon 6A is used by non muscle and undifferentiated muscle cells (myoblasts) while exon 6B is exclusively used in differentiated skeletal muscle cells. A complex array of cis acting sequence elements are involved in the regulation of this alternative splicing process. Transfection assays of quail muscle cells in culture were used to define these cis acting elements. We show that, in undifferentiated muscle cells, exon 6B is skipped as a result of a negative control on its selection while exon 6A is spliced as a default choice. We provide evidence that this negative control involves a secondary structure of the primary transcript around the 5' end of exon 6B as well as intronic sequence elements located between the branch point and the acceptor splice site of exon 6B. In differentiated muscles, both exons are accessible to the splicing machinery and the preferential use of exon 6B depends on the existence of a competition between the two exons for the selection of the flanking splice sites. In particular, we show that the donor splice site of exon 6A is a weak splice site while the branch point associated with exon 6B is a strong branch point.

Alternative Splicing↗

PubMed1997

Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing.

We report the isolation and characterization of a third isoform of placenta growth factor (PlGF), generated by alternative splicing of its RNA transcript. This novel form of PlGF, PlGF-3, was cloned by the polymerase chain reaction technique using a human cDNA library prepared from the terminal placental tissue. PlGF-3 contains an in-frame insertion of 72-amino acids near the C-terminal portion of PlGF-1. Southern blot analysis revealed that a single gene encoded PlGF-2 and PlGF-3. Nucleic acid sequence analysis found the insertion of 216 nucleotides of PlGF-3 between exon 4 and exon 5 of the PlGF gene. Northern blot and tissue distribution studies discovered two mRNA species for PlGF-3, which were both uniquely expressed in the placenta. Transient expression of PlGF-3 cDNA in mammalian cells showed PlGF-3 was detected in the conditioned medium as both dimers and monomers. Unlike PlGF-2, PlGF-3 lacked heparin-binding affinity. Thus, alternative splicing of PlGF RNA produces at least three polypeptides with different secretion pattern, heparin-binding affinity and dimerization properties.

Alternative Splicing↗

PubMed2004

Alternative splicing in the first alpha-helical region of the Rab-binding domain of Rim regulates Rab3A binding activity: is Rim a Rab3 effector protein during evolution?

Rim1 and Rim2 were originally described as specific Rab3A-effector proteins involved in the regulation of secretory vesicle exocytosis. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs. Although alternative splicing in the RBD1 of Rim is known to produce long and short forms of RBD (named Rim1 and Rim1Delta56-105, and Rim2(+40A) and Rim2, respectively), with the long form of Rim1 and short form of Rim2 being dominant in mouse brain, the physiological significance of the alternative splicing in RBD1 has never been elucidated. In the present study I discovered that alternative splicing in Rim RBD1 alters Rab3A binding affinity to Rims, and found that insertion of 40 amino acids into the RBD1 of Rim2 (i.e. Rim2(+40A)) dramatically reduced its Rab3A binding activity (more than a 50-fold decrease in affinity). Similarly, Rim1Delta56-105 exhibited higher affinity binding to Rab3A than the long form of Rim1. Expression of the short forms of the Rim RBD in PC12 cells co-localized well with endogenous Rab3A, whereas expression of the long forms of the Rim RBD in PC12 cells resulted in cytoplasimc and nuclear localization. Moreover, I found that Caenorhabditis elegans Rim/UNC-10 (ce-Rim) and Drosophila Rim (dm-Rim) do not interact with ce-Rab3 and dm-Rab3, respectively, indicating that the Rab3-effector function of Rim has not been retained during evolution. Based on these findings, I propose that the Rab3A-effector function of Rim during secretory vesicle exocytosis is limited to the short form of the mammalian Rim RBD alone.

Alternative Splicing↗

PubMed1996

Purification and characterization of the soluble interleukin-6 receptor from human plasma and identification of an isoform generated through alternative splicing.

The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein 1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane-bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity-purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.

Alternative Splicing↗

PubMed1998

Control of alternative splicing of potassium channels by stress hormones.

Many molecular mechanisms for neural adaptation to stress remain unknown. Expression of alternative splice variants of Slo, a gene encoding calcium- and voltage-activated potassium channels, was measured in rat adrenal chromaffin tissue from normal and hypophysectomized animals. Hypophysectomy triggered an abrupt decrease in the proportion of Slo transcripts containing a "STREX" exon. The decrease was prevented by adrenocorticotropic hormone injections. In Xenopus oocytes, STREX variants produced channels with functional properties associated with enhanced repetitive firing. Thus, the hormonal stress axis is likely to control the excitable properties of epinephrine-secreting cells by regulating alternative splicing of Slo messenger RNA.

Adrenal Medulla↗

PubMed2006

AU-rich elements and alternative splicing in the beta-catenin 3'UTR can influence the human beta-catenin mRNA stability.

Beta-catenin, the central player of the Wnt signaling cascade, is a well-known oncogene. The regulation of beta-catenin protein stability has been studied extensively while other mechanisms that control cellular levels of beta-catenin have hardly been addressed. In this study, we show that there are three beta-catenin mRNA splice variants that differ solely in their 3'-untranslated region (3'UTR) due to alternative splicing or retaining of an intron. The three isoforms were found to be ubiquitously expressed though in different quantities. Upon induction of the beta-catenin protein in peripheral blood mononuclear leukocytes (PBMC), the beta-catenin mRNA is induced in an isoform-specific manner. All three variants occur in the cytoplasm and contribute to the synthesis of beta-catenin acting as a transcriptional coactivator but have different cytoplasmic stabilities in Hela cells. AU-rich elements (AREs), sequence elements implicated in the regulation of mRNA stability, are found in each of the three transcripts. Surprisingly, the AREs contribute to stabilization of the beta-catenin mRNA transcripts in a splicing-dependent manner. The isoform most affected is the one found to be most induced when beta-catenin protein accumulates. These results suggest that alternative splicing and AREs can act together in regulating beta-catenin mRNA stability and thereby provide a step of controlling the cellular beta-catenin concentration.

3' Untranslated Regions↗

PubMed2002

Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns.

Adenosine deaminase that acts on RNA -2 (ADAR2) is a member of a family of vertebrate genes that encode adenosine (A)-to-inosine (I) RNA deaminases, enzymes that deaminate specific A residues in specific pre-mRNAs to produce I. Known substrates of ADAR2 include sites within the coding regions of pre-mRNAs of the ionotropic glutamate receptors, GluR2-6, and the serotonin receptor, 5HT2C. Mammalian ADAR2 expression is itself regulated by A-to-I editing and by several alternative splicing events. Because the biological consequences of ADAR2 function are significant, we have undertaken a phylogenetic comparison of these features. Here we report a comparison of cDNA sequences, genomic organization, editing site sequences and patterns of alternative splicing of ADAR2 genes from human, mouse, chicken, pufferfish and zebrafish. Coding sequences and intron/exon organization are highly conserved. All ADAR2 genes show evidence of transcript editing with required sequences and predicted secondary structures very highly conserved. Patterns and levels of editing and alternative splicing vary among organisms, and include novel N-terminal exons and splicing events.

Adenosine Deaminase↗

PubMed2000

A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing.

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing. Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A. brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.

Alternative Splicing↗

PubMed1993

Alternative splicing of the mRNA encoding the alpha subunits of the G(o) GTP-binding protein during brain development and in neuronal cell lines.

The RNA encoding the alpha subunit of the G(o) GTP-binding protein is alternatively spliced in some tissues to produce mRNAs encoding two distinct forms of the protein which differ at the carboxyl terminus. This alternative splicing process is observed at all stages during development of the rat brain and heart and in some but not all neuronal cell lines. These effects are discussed both in terms of the regulation of the alternative splicing event and the functional role(s) of the two forms of the protein which it produces.

3T3 Cells↗

PubMed2004

Alternative splicing of Bcl-2-related genes: functional consequences and potential therapeutic applications.

Apoptosis is a morphologically distinct form of cell death. It is executed and regulated by several groups of proteins. Bcl-2 family proteins are the main regulators of the apoptotic process acting either to inhibit or promote it. More than 20 members of the family have been identified so far and most have two or more isoforms. Alternative splicing is one of the major mechanisms providing proteomic complexity and functional diversification of the Bcl-2 family proteins. Pro- and anti-apoptotic Bcl-2 family members should function in harmony for the regulation of the apoptosis machinery, and their relative levels are critical for cell fate. Any mechanism breaking down this harmony by changing the relative levels of these antagonistic proteins could contribute to many diseases, including cancer and neurodegenerative disorders. Recent studies have shown that manipulation of the alternative splicing mechanisms could provide an opportunity to restore the proper balance of these regulator proteins. This review summarises current knowledge on the alternative splicing products of Bcl-2-related genes and modulation of splicing mechanisms as a potential therapeutic approach.

Alternative Splicing↗