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Results for “splicing variant”

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PubMed1999

Immunohistochemical localization of the somatostatin sst2(b) receptor splice variant in the rat central nervous system.

Somatostatin is a neuromodulator in the mammalian CNS. To date, genes for at least five different somatotrophin release inhibiting factor receptors, termed sst1-sst5, have been cloned. The rat sst2 receptor exists in two splice variants, sst(alpha)a) and sst2(b), which differ in their carboxy-termini. When heterologously expressed in Chinese hamster ovary-K1 cells, these splice variants show little difference in their operational characteristics. Recently, the distribution of the sst2(a) receptor was documented, yet at present no data are available about the distribution of the sst2(b) receptor in the CNS. Here, we present the characterization of a novel polyclonal anti-peptide antibody that is selective for the sst2(b) receptor splice variant. The antibody was raised against the unique intracellular carboxy-terminal portion of the receptor protein. Using this affinity-purified antibody in western blotting experiments, the sst2(b) receptor expressed in Chinese hamster ovary-K1 cells was shown to be a glycoprotein with a molecular weight centred at about 85,000. The antibody showed no cross-reactivity to any of the recombinant human sst1-5 receptors, the rat sst2(a) receptor or wild-type Chinese hamster ovary-K1 cells. Employing immunohistochemistry, we investigated the distribution of the sst2(b) receptor in the brain and spinal cord of adult rats. A distinct distribution was found throughout the rostrocaudal axis of the CNS. Somatodendritic as well as axonal staining was observed. Somatodendritic labelling was particularly obvious in the olfactory bulb, cerebral cortex, hippocampal formation, mesencephalic trigeminal nucleus and cerebellum, as well as in cranial and spinal motor areas. The results show that the distribution of the sst2(b) receptor partially overlaps with that of the sst2(b) receptor, although there were differences in a number of brain areas. The location of the sst2(b) receptor implies that it may mediate a modulatory role of somatostatin inhibitory releasing factor on sensory as well as motor functions.

Animals↗

PubMed2001

Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos.

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.

Active Transport, Cell Nucleus↗

PubMed1998

Multiple amphiphysin II splice variants display differential clathrin binding: identification of two distinct clathrin-binding sites.

Amphiphysin I and II are nerve terminal-enriched proteins that display src homology 3 domain-mediated interactions with dynamin and synaptojanin. It has been demonstrated that the amphiphysins also bind to clathrin, and we have proposed that this interaction may help to target synaptojanin and dynamin to sites of synaptic vesicle endocytosis. To understand better this potential functional role, we have begun to characterize clathrin-amphiphysin interactions. Using PCR from adult human cortex cDNA, we have cloned a number of amphiphysin II splice variants. In in vitro binding assays, the amphiphysin II splice variants display differential clathrin binding and define a 44-amino acid region mediating the interaction. Amphiphysin II truncation and deletion mutants identify two distinct clathrin-binding domains within this region: one with the sequence LLDLDFDP, the second with the sequence PWDLW. Both domains are conserved in amphiphysin I, and saturation binding analysis demonstrates that both sites bind clathrin with approximately equal affinity. The elucidation of clathrin as a splice-specific binding partner for amphiphysin II begins to address the potential functional role(s) for the multiple amphiphysin II splice variants and further supports an important function for clathrin-amphiphysin interactions in protein targeting during endocytosis.

Alternative Splicing↗

PubMed2005

Identification and functional characterization of an alternative splice variant within the fourth exon of human nanog.

Nanog, a homeodomain (HD) transcription factor, plays a critical role in the maintenance of embryonic stem (ES) cell self-renewal. Here, we report the identification of an alternatively-spliced variant of nanog. This variant lacked a stretch of amino acids (residues 168-183) located between the HD and tryptophan-repeat (WR) of the previously-reported full length sequence, suggesting that the deleted sequence functions as a linker and possibly affects the flexibility of the C-terminal transactivation domain relative to the DNA binding domain. Expression of mRNA encoding the splice variant, designated as nanog-delta 48, was much lower than that of the full length version in human ES cells. The ratio of nanog-delta 48 transcript to full length transcript increased, however, in multipotent adult progenitor cells. EMSA analysis revealed that both forms of Nanog were able to bind a nanog binding sequence with roughly the same affinity. A reporter plasmid assay also showed that both variants of nanog modestly repressed transactivation of gata-4, whose expression is proposed to be inhibited by nanog, with comparable potency. We conclude that, despite the difference in primary structure and expression pattern in various stem cells, the alternatively-spliced variant of Nanog has similar activity to that of the full length version.

Alternative Splicing↗

PubMed2006

Splice variant 3, but not 2 of receptor protein-tyrosine phosphatase sigma can mediate stimulation of insulin-secretion by alpha-latrotoxin.

Alpha-latrotoxin (alpha-LTX) binds to several cell surface receptors including receptor protein-tyrosine phosphatase sigma (RPTPsigma). Here we demonstrate that transient overexpression of the short splice variant 3 conferred alpha-LTX induced secretion to hamster insulinoma (HIT-15) cells. In contrast, the long splice variant 2 containing four additional extracellular fibronectin-III domains was inactive in secretion or in a single cell assay. Toxin-sensitive (MIN6) and toxin-insensitive (HIT-T15) insulinoma cell lines as well as PC12 cells expressed similar amounts of endogenous short RPTPsigma splice variant suggesting that this receptor does not play a role for toxin-sensitivity.

Animals↗

PubMed1992

Oligonucleotide probes detect splicing variants in situ in Drosophila embryos.

We describe a method for the in situ detection of specific splicing variants. The method is based on the use of antisense oligonucleotides designed to span splice junctions labelled with digoxigenin by terminal transferase tailing. We find that the spatial patterns of Ubx splicing variants Ia and IIa are similar in early embryos, but differ in late embryos. Variant IVa is only detected in the CNS (ps6) at stages 16 and 17. We also present evidence indicating that the first splicing event is cotranscriptional.

Alternative Splicing↗

PubMed2004

Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

Alternative Splicing↗

PubMed2003

Cell surface expression of NR1 splice variants and NR2 subunits is modified by prenatal ethanol exposure.

N-methyl-D-aspartate receptor dysfunction has been strongly suggested to link with the abnormalities seen in fetal alcohol syndrome. Thus, the effects of prenatal ethanol exposure on the total expression of NR1 splice variants and the cell surface expression of both NR1 and NR2 subunits in brain were investigated in rats. Western blot studies of membrane homogenates from cerebral cortices at postnatal days 1 through 21 indicate that prenatal ethanol treatment does not alter total NR1 expression or differential expression of NR1 splice variants during development. However, immunoprecipitation studies using PSD95 suggest that both C2'-terminal variants and NR2A subunits at the cortical postsynaptic membrane of postnatal day 21 were significantly reduced after prenatal ethanol treatment. Moreover, C1-terminal variants were decreased in both pair-fed and ethanol-treated groups, while no significant differences in the levels of total NR1 subunits, NR1 splice variants containing the N- or C2-terminal cassettes, or NR2B subunits were observed. Thus, these results suggest that prenatal exposure to ethanol may influence neuronal function by selective regulation of expression of C2'-terminal variants and NR2A subunits at the cell surface.

Aging↗

PubMed2003

Gene structure-based splice variant deconvolution using a microarray platform.

MOTIVATION: Alternative splicing allows a single gene to generate multiple mRNAs, which can be translated into functionally and structurally diverse proteins. One gene can have multiple variants coexisting at different concentrations. Estimating the relative abundance of each variant is important for the study of underlying biological function. Microarrays are standard tools that measure gene expression. But most design and analysis has not accounted for splice variants. Thus splice variant-specific chip designs and analysis algorithms are needed for accurate gene expression profiling. RESULTS: Inspired by Li and Wong (2001), we developed a gene structure-based algorithm to determine the relative abundance of known splice variants. Probe intensities are modeled across multiple experiments using gene structures as constraints. Model parameters are obtained through a maximum likelihood estimation (MLE) process/framework. The algorithm produces the relative concentration of each variant, as well as an affinity term associated with each probe. Validation of the algorithm is performed by a set of controlled spike experiments as well as endogenous tissue samples using a human splice variant array.

Algorithms↗

PubMed2006

A splice variant of the human CCA-adding enzyme with modified activity.

The human CCA-adding enzyme (tRNA nucleotidyltransferase) is an essential enzyme that catalyzes the addition of the CCA terminus to the 3' end of tRNA precursors, a reaction which is a fundamental prerequisite for mature tRNAs to become aminoacylated and to participate in protein biosynthesis. To date only one form of this enzyme has been identified in humans. Here, we describe the sequence and activity of a splice variant of the human CCA-adding enzyme identified in public cDNA databases. The in silico analyses performed on this splice variant indicate that there is conservation of the alternative splice donor site among species and indicate that it seems to be used in vivo. Moreover, the recombinantly expressed protein is active in vitro and accepts tRNA transcripts as substrates incorporating the dinucleotide sequence CC to their 3' end, in contrast to the activity of the full length enzyme. These findings strongly suggest that the splice variant of the human CCA-adding enzyme is expressed in the cell although the in vivo function remains unclear.

Adenosine Triphosphate↗

PubMed2005

Co-assembly of two GluR6 kainate receptor splice variants within a functional protein complex.

Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.

Alternative Splicing↗

PubMed2006

Ca(v)3.1 splice variant expression during neuronal differentiation of Y-79 retinoblastoma cells.

A decrease in transient-type calcium channel current, Ca(v)3.1 protein and the mRNA encoding these channels has been reported during differentiation of human retinoblastoma cells. In this study, we examined splice variants of Ca(v)3.1 before and after neuronal differentiation of the Y-79 retinoblastoma cell line to investigate the potential contribution of Ca(v)3.1 to Y-79 differentiation. In Ca(v)3.1, alternative splicing induces variations in three cytoplasmic regions, e.g. the link between domains II and III (producing isoforms e+ and e-), the link between domains III and IV (producing isoforms a, b, ac and bc) and the carboxy terminal region (producing isoforms f and d). Our results demonstrate that Ca(v)3.1e was not expressed in either undifferentiated or differentiated retinoblastoma cells. Splice variants Ca(v)3.1ac; Ca(v)3.1bc and Ca(v)3.1b were all identified in undifferentiated retinoblastoma cells, while expression of these variants in differentiated cells was restricted to the Ca(v)3.1bc isoform. The carboxy terminal variant Ca(v)3.1f is expressed independently of the differentiation status of retinoblastoma cells with or without Ca(v)3.1d. Examination of the functional contribution of Ca(v)3.1 protein to Y-79 cell differentiation revealed that in Y-79 cells transfected with Ca(v)3.1 antisense oligodeoxynucleotides, knockdown of Ca(v)3.1 did not alter the time-course of differentiation or neuritogenesis. The changes in Ca(v)3.1 splice variants were not required for the initiation of differentiation but may be associated with tissue-specific expression or localized alterations in Ca(2+) signaling that are essential for establishment of the mature differentiated phenotype.

Alternative Splicing↗

PubMed1998

Novel splice variants of human tenascin-C mRNA identified in normal and bullous keratopathy corneas.

PURPOSE: Pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas accumulate an extracellular matrix glycoprotein tenascin-C (TN-C), an important modulator of cell adhesion and migration. Here, the purpose was to identify specific TN-C mRNA splice variants in normal and PBK/ABK human corneas. METHODS: Conventional and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) with primers to alternatively spliced (insertional) and constitutive fibronectin type II-like repeats of TN-C was used. Splice variants were identified by cloning and sequencing of RT-PCR products or by Southern blot analysis. RESULTS: The majority of corneal TN-C mRNA species corresponded to relatively small forms of the protein. Four previously unidentified TN-C mRNA splice variants were found in normal and PBK/ABK corneas that contained insertional repeats A1+A2+B+D, A1+A2+D, A1+B+D, or A1+D. Variants with insertional repeats A1+A2 or A1, previously described in mouse and rat, were also identified in human corneas. Semiquantitative RT-PCR showed that novel TN-C mRNA variants were dramatically elevated in PBK/ABK compared to normal corneas. CONCLUSION: TN-C protein was found in PBK/ABK but not in normal corneas; however, both normal and diseased corneas contained mRNA for 15 different TN-C isoforms. PBK/ABK corneas had elevated levels of six relatively small TN-C mRNA variants including five novel ones. These specific isoforms may adversely affect adhesion and migration of corneal cells thus contributing to the exacerbation of PBK/ABK.

Alternative Splicing↗

PubMed2003

Expression of a splice variant of choline acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat.

A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component.

Acetylcholine↗

PubMed2006

Transcriptional expression of survivin and its splice variants in endometriosis.

Survivin is a novel inhibitor of apoptosis (IAP), and the two splice variants of survivin (survivin-2B and survivin-EX3) have been identified. Gene expression levels of survivin, survivin-2B and survivin-EX3 in 56 ectopic (16 peritoneal red and 16 peritoneal black lesions and 24 ovarian endometriomata) and 13 eutopic endometrial tissues surgically obtained from 42 women with endometriosis (group A) were compared with those in 16 control eutopic endometrium from 16 women without endometriosis (group B) by quantitative RT-PCR analysis. Survivin mRNA expression levels in ectopic endometriotic tissues were significantly higher than those in eutopic endometrium of groups A and B over the whole cycle. Red peritoneal lesions had higher gene expression levels of survivin than black lesions. In contrast, all tissue samples examined showed relatively lower gene expression levels of survivin-2B and survivin-EX3. No cyclic variation was found in survivin and the two splice variants, both in ectopic and in eutopic endometrium. Although there was no significant difference in the ratio of survivin-2B/survivin between ectopic and eutopic endometrium, the ratio of survivin-EX3/survivin in peritoneal endometriotic lesions was significantly higher than that of eutopic endometrium of groups A and B. These results suggest that survivin and survivin-EX3 may be closely linked to escape from apoptosis and the development of endometriosis.

Adult↗

PubMed1997

Estrogen receptor messenger RNA splice variants are not involved in antiestrogen resistance in sublines of MCF-7 human breast cancer cells.

Development of resistance to tamoxifen is a serious problem in treatment of breast cancer patients. Although the mechanisms for development of resistance are unclear, an altered expression of alternatively spliced estrogen receptor (ER) mRNA has been suggested to be involved. We have looked for differential expression of ER splice variants lacking exon 2 (ERdeltaE2), exon 3 (ERdeltaE3), exon 4 (ERdeltaE4), exon 5 (ERdeltaE5), exon 7 (ERdeltaE7), and exons 4 and 7 (ERdeltaE4, 7) in the human breast cancer cell line MCF-7 and 10 ER-positive MCF-7 sublines resistant to the antiestrogens tamoxifen, ICI 164,384 or ICI 182,780. No major differences in the expression were demonstrated between MCF-7 cells and resistant cells, indicating that ER splice variants are not involved in antiestrogen resistance in this model system. Furthermore, despite a high mRNA level of some of the ER splice variants, no corresponding proteins could be detected using Western blot analysis.

Antineoplastic Agents, Hormonal↗

PubMed2004

Versican splice variant messenger RNA expression in normal human Achilles tendon and tendinopathies.

OBJECTIVES: Versican is the principal large proteoglycan expressed in mid-tendon, but its role in tendon pathology is unknown. Our objective was to define the expression of versican isoform splice variant messenger ribonucleic acid (mRNA) in normal Achilles tendons, in chronic painful tendinopathy and in ruptured tendons. METHODS: Total RNA isolated from frozen tendon samples (normal n = 14; chronic painful tendinopathy n = 10; ruptured n = 8) was assayed by relative quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for total versican, versican variants V0, V1, V2, V3 and type I collagen alpha1 mRNA, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Differences between sample groups were tested by Wilcoxon statistics. RESULTS: Painful and ruptured tendons showed a significant decrease (median 2-fold) in the expression of versican mRNA, in contrast to an increased expression (median 8-fold) of type I collagen alpha1 mRNA in painful tendons. Versican splice variants V0 and V1 mRNA were readily detected in normal samples, V3 levels were substantially lower, and V2 levels were more variable. Each of V1, V2 and V3 mRNA showed significant decreases in expression in painful and ruptured tendons, but V0 was not significantly changed. CONCLUSIONS: Changes in versican expression relative to that of collagen, and alterations in the balance of versican splice variants, may contribute to changes in matrix structure and function in tendinopathies.

Achilles Tendon↗

PubMed1993

Homomeric assemblies of NMDAR1 splice variants are sensitive to ethanol.

Studies have shown that the neuronal NMDA receptor is a target for the actions of ethanol. Recently, a number of subunits of the NMDA receptor have been cloned and functionally expressed in various combinations. We have expressed four splice variants of the NMDAR1 subunit in Xenopus oocytes, and find that homomeric assemblies of this subunit, in the absence of other subunits, exhibit ethanol sensitivity comparable to that seen in neurons. In the presence of calcium, the reduction of total current was greatest in the NMDAR1-LL splice variant, and was significantly less in the NMDAR1-SS variant. The increased sensitivity of NMDAR1-LL may be attributed to a particularly sensitive slow current 'hump' which is more pronounced in NMDAR1-LL than in NMDAR1-SS. The reduction of NMDA-evoked current by ethanol was significantly different when calcium was replaced by barium in the external medium. In this case, the slow current hump was significantly reduced, current through NMDAR1-LL was less reduced by ethanol, and the percent reduction of NMDAR1-LL and NMDAR1-SS currents was similar. NMDA-evoked currents in heteromeric receptors formed by coinjection of the mouse epsilon-1 subunit with the NMDAR1 splice variants responded to ethanol similarly to homomeric assemblies.

Animals↗