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PubMed2002

Identification and characterization of a novel human cathepsin L splice variant.

Human cathepsin L (hCATL) has been implicated in a variety of physiological and pathological processes. It was hitherto known to be encoded by four mRNA species, namely hCATL A, AI, AII and hCATL B, differing in their 5' untranslated regions (UTRs). Of these, hCATL A, AI and AII are produced by the alternative splicing of the same primary transcript. HCATL AI and hCATL AII, lack 27 and 90 bases, respectively, from the 3' end of exon 1 of hCATL A. The present study describes the identification of a new splice variant hCATL AIII, which similarly lacks 145 bases from the 3' end of exon 1 of hCATL A. It is produced by the splicing out of 136-280 bases of the first exon in addition to intron 1 of hCATL A, which together serve as an intron for hCATL AIII. HCATL AIII was observed to be the most abundant splice variant in five different human cell lines. In vitro transcription coupled translation studies revealed that hCATL AIII is translated with 4.4-, 3.9- and 1.6-fold higher efficiency as compared to hCATL A, AI and AII, respectively. These results were further confirmed by measuring the enzymatic activities of the in vitro translated products. Cloning of hCATL AIII UTR upstream to luciferase reporter gene resulted in a 3.75-fold higher expression of the reporter gene as compared to the luciferase construct containing UTR of hCATL A. Thus, we have identified a novel human cathepsin L splice variant, hCATL AIII, which is most abundant in human cell lines and is translated with highest efficiency. Our results demonstrate either the presence of a positive or absence of a negative cis-acting regulatory element(s) in the UTR of hCATL AIII that is sufficient to confer translational advantage to a heterologous mRNA. The predominance of this most efficiently translated splice variant in malignant cells suggests that it plays a key role in the over-expression of human cathepsin L in cancer.

3T3 Cells↗

PubMed2005

Identification of an N-terminal TRPC2 splice variant which inhibits calcium influx.

TRPC2 is a member of the transient receptor potential (TRP) superfamily of Ca2+-permeable channels expressed in nonexcitable cells. TRPC2 is involved in a number of physiological processes including sensory activation of the vomeronasal organ, sustained Ca2+ entry in sperm, and regulation of calcium influx by erythropoietin. Here, a new splice variant of TRPC2, called "Similar to mouse TRPC2" (smTRPC2), was identified consisting of 213 amino acids, largely coincident with the N-terminus of TRPC2 clone 17. This splice variant lacks all six TRPC2 transmembrane domains and the calcium pore. Expression of smTRPC2 was found in all tissues examined by RT-PCR and in primary erythroid cells by RT-PCR and Western blotting. Confocal microscopy of CHO-S cells transfected with TRPC2 clone 14 and smTRPC2 demonstrated that TRPC2 clone 14 and smTRPC2 both localize at or near the plasma membrane and in the perinuclear region. Cell surface localization of TRPC2 was confirmed with biotinylation, and was not substantially affected by smTRPC2 expression. Coassociation of TRPC2 c14 and alpha with smTRPC2 was confirmed by immunoprecipitation. To examine the functional significance of smTRPC2 expression, a CHO-S model was used to study its effect on calcium influx stimulated by Epo through TRPC2. Single CHO-S cells which express transfected Epo-R were identified by detection of green fluorescent protein (GFP). Cells that express transfected TRPC2 c14 or alpha were identified by detection of blue fluorescent protein (BFP). [Ca]i was quantitiated with Fura Red fluorescence using digital video imaging. Epo stimulated calcium influx through TRPC2 isoforms c14 and alpha, which was inhibited by coexpression of smTRPC2. These data demonstrate that a short splice variant of TRPC2 exists in many cell types, which associates with and modifies the activity of functional TRPC2 splice variants.

Alternative Splicing↗

PubMed2001

Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction.

A kinetic reverse transcription-polymerase chain reaction (RT-PCR)-based assay is described that can discriminate and quantitate differentially spliced mRNAs. This assay should be generally applicable for high-throughput quantitation of differentially spliced transcripts. The utility of this method was assessed for spliced transcripts encoded by the human Na+-K+-2Cl- cotransporter gene hNKCC1. Evidence is presented that the NKCC1 isoform of the human Na+-K+-2Cl- cotransporter is differentially spliced analogous to that recently described for the mouse Na+-K+-2Cl- cotransporter gene BSC2. The nucleotide sequences of the two human splice variants predict Na+-K+-2Cl- cotransporter proteins differing only in length. Stable transfectants expressing these human splice variants, designated NKCC1a or NKCC1b, were constructed. Both splice variants produce functional Na+-K+-2Cl- cotransporters in vivo. The abundance of NKCC1 mRNA and patterns of differential splicing in 10 different tissue types and three cell lines were quantitated using the kRT-PCR assay. The results showed that the total amount of NKCC1 mRNA varied by more than 30-fold in the human tissues and cell lines examined. The ratio of NKCC1a/NKCC1b varied nearly 70-fold among these same tissues and cell lines suggesting that differential splicing of the NKCC1 transcript may play a regulatory role in human tissues.

Alternative Splicing↗

PubMed2007

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro.

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.

Alternative Splicing↗

PubMedDate not supplied

Expression of crisp-1 mRNA splice variants in the rat epididymis, and comparative analysis of the rat and mouse crisp-1 gene regulatory regions.

The rat Crisp-1 gene encodes Protein DE (acidic epididymal glycoprotein; AEG), a glycoprotein secreted by the epididymal epithelium that associates with maturing sperm and has been implicated in the process of sperm-egg fusion. Previous characterization of the Crisp-1 messenger RNA in the rat epididymis has demonstrated the presence of 3 splice variants (Klemme et at, 1999). This study was undertaken to determine if expression of the Crisp-1 splice variants in the rat epididymis is region-specific and correlates with the region-specific pattern of synthesis of the D and E forms of the Crisp-1 protein. Expression of each of the splice variants was shown by RNase protection assays to be under the control of androgens, but they are not differentially regulated either within the epididymal segments or along the length of the organ. The reported structure of the mouse Crisp-1 gene does not include an exon that is equivalent to the rat exon 1, suggesting that the rat splice variants cannot exist in the mouse and may be specific to the rat. Furthermore, the mouse transcription start site is situated in a different region of the gene than in the rat. In this study, a comparison of the mouse and rat genes in the region flanking the mouse exon 1 and the rat exon 2 (within the rat intron 1) shows greater than 80% sequence identity, including the conservation of several putative androgen receptor binding sites. In addition, the rat gene is shown to have a corrupted TATA box in intron 1 that corresponds to the TATA box located in the mouse gene. These observations explain the preferential transcription for the mouse gene in this region, while the predominant start site for the rat gene is 5' of the upstream exon 1. Although an exon corresponding to the rat exon 1 has not been found in the mouse gene, reverse transcription-polymerase chain reaction experiments using mouse epididymal RNA suggest that such an exon exists in the mouse gene and is transcribed at low frequency.

Alternative Splicing↗

PubMed2000

Cloning and characterization of two splice variants of human phosphodiesterase 11A.

Phosphodiesterase 11A (PDE11A) is a recently identified family of cAMP and cGMP hydrolyzing enzymes. Thus far, a single splice variant designated as PDE11A1 has been reported. In this study, we identify and characterize two additional splice variants of PDE11A, PDE11A2 and PDE11A3. The full-length cDNAs are 2,141 bp for PDE11A2 and 2205 bp for PDE11A3. The ORF of PDE11A2 predicts a protein of 576 aa with a molecular mass of 65.8 kDa. The ORF of PDE11A3 predicts a protein of 684 aa with a molecular mass of 78.1 kDa. Comparison of the PDE11A2 sequence with that of PDE11A1 indicates an additional 86 aa at the N terminus of PDE11A2. Part of this sequence extends the potential cGMP binding region (GAF domain) present in PDE11A1. Compared with PDE11A2, PDE11A3 has an additional 108 N-terminal amino acids. Sequence analysis of PDE11A3 indicates the presence of another GAF domain in this region. This diversification of regulatory sequences in the N-terminal region of PDE11A splice variants suggests the interesting possibility of differential regulation of these enzymes. Recombinant PDE11A2 and -A3 proteins expressed in the Baculovirus expression system have the ability to hydrolyze both cAMP and cGMP. The K(m) values for cAMP hydrolysis are 3.3 microM and 5.7 microM for PDE11A2 and PDE11A3, respectively. The K(m) values for cGMP hydrolysis are 3.7 microM and 4.2 microM for PDE11A2 and PDE11A3, respectively. Both PDEs showed a V(max) ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an IC(50) of 1.8 microM, and to zaprinast, with an IC(50) of 28 microM. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with IC(50) values of 0.82 and 5 microM for dipyridamole and zaprinast, respectively.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

Distribution of two splice variants of the glutamate transporter GLT-1 in rat brain and pituitary.

We have performed immunocytochemistry on rat brains using a highly specific antiserum directed against the originally described form of the glutamate transporter GLT-1 (referred to hereafter as GLT-1alpha), and another against a C-terminal splice variant of this protein, GLT-1B. Both forms of GLT-1 were abundant in rat brain, especially in regions such as the hippocampus and cerebral cortex, and macroscopic examination of sections suggested that both forms were generally regionally coexistent. However, disparities were evident; GLT-1alpha was present in the intermediate lobe of the pituitary gland, whereas GLT-1B was absent. Similar marked disparities were also noted in the external capsule, where GLT1A labeling was abundant but GLT-1B was only occasionally encountered. Conversely, GLT-1B was more extensively distributed, relative to GLT-1alpha, in areas such as the deep cerebellar nuclei. In most regions, such as the olfactory bulbs, both splice variants were present but differences were evident in their distribution. In cerebral cortex, patches were evident where GLT-1B was absent, whereas no such patches were evident for GLT-1alpha. At high resolution, other discrepancies were evident; double-labeling of areas such as hippocampus indicated that the two splice variants may either be differentially expressed by closely apposed glial elements or that the two splice variants may be differentially targeted to distinct membrane domains of individual glial cells.

Alternative Splicing↗

PubMed1993

Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor.

We have determined the gene structure for the NMDA receptor subunit gene NMDAR1. We found eight splice variants that arise from different combinations of a single 5' terminal exon insertion and three different 3' terminal exon deletions, relative to NMDAR1. We analyzed the modulation by Zn2+ of currents through homomeric receptors assembled from these splice variants and found that, in addition to its well-known inhibitory effect at high concentrations, Zn2+ potentiates agonist-induced currents at submicromolar concentrations (EC50 = 0.50 microM). This potentiation is observed only with a subset of NMDAR1 splice variants that show additional differences in pharmacological properties. Zn2+ potentiation is rapidly reversible, noncompetitive with either glutamate or glycine, and voltage independent. Zn2+ potentiation is mimicked by Cd2+, Cu2+, and Ni2+, but not by Mn2+, Co2+, Fe3+, Sn2+, or Hg2+. Our results suggest a possible role for Zn2+ as a positive modulator of NMDA receptors in certain regions of the brain.

Animals↗

PubMed1992

The two splice variants of collagen XII share a common 5' end.

The short splice variant of collagen XII is composed of 1960 amino acid residues. This polypeptide contains the same signal peptide and the same carboxy-terminus as the long splice variant, but lacks an internal fragment of 1164 amino acid residues. Thus, the two variants of collagen XII are not created by the use of two different transcription initiation sites as generally assumed, but result from the inclusion or skipping of several exons located within the collagen XII gene.

Amino Acid Sequence↗

PubMed2006

Analysis of prostate-specific membrane antigen splice variants in LNCap cells.

The prostate-specific membrane antigen (PSMA), a product of the folate hydrolase (FOLH1) gene, is highly expressed as a largely extracellular membrane-anchored protein in malignant prostate tissues and in nonprostatic tumor neovasculature. Treatment of prostate cancer LNCap cells with spliceswitching oligonucleotides (SSOs) modulated splicing of FOLH1 pre-mRNA from the full-length PSMA splice variant to three splice variants: the cytoplasmic PSM', alternatively spliced at exon 1, and the previously unexamined PSMADelta6 and PSMADelta18 variants, which lack exons 6 and 18, respectively. Application of SSOs decreased membrane PSMA levels and increased PSM', PSMADelta6, and PSMADelta18 transcripts. As a result, PSM' protein was translocated to the cytoplasm, and switching to PSMADelta6 and PSMADelta18 downregulated PSMA expression. NAALADase assays showed that PSM' retained enzymatic activity. PSMADelta6 and PSMADelta18 were not active, presumably due to a change in a reading frame that eliminated the NAALDase active site or the dimerization domain or both in these proteins.

Alternative Splicing↗

PubMed2003

Selective decrease in NR1 subunit splice variant mRNA in the hippocampus of Pb2+-exposed rats: implications for synaptic targeting and cell surface expression of NMDAR complexes.

We have previously shown that exposure to environmentally relevant levels of Pb(2+) during brain development decreases the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) and NR2A genes in the hippocampus of young adult rats and was associated with deficits in hippocampal LTP and spatial learning [Neuroscience 99 (2000) 233-242]. In the present study, we demonstrate that the lower levels of NR1 subunit mRNA expressed in the Pb(2+)-exposed hippocampus are principally due to decreased levels of the NR1-4 and NR1-2 splice variants. These changes were present in the absence of changes in GluR1, PSD-95 and alphaCaMKII gene expression. A unique characteristic of these splice variants is that they lack the C1 cassette. Further, these splice variants have been shown to impart the highest cell surface expression, PKC potentiation and calcium kinetics to NMDAR complexes. Our present findings indicate that Pb(2+)-induced changes in NR1 subunit splice variant mRNA expression in the hippocampus may provide a mechanism by which Pb(2+)-exposure can modify NMDAR-mediated calcium signaling and influence the degree of synaptic plasticity.

Alternative Splicing↗

PubMed2006

Identification of a novel pituitary-specific chicken gonadotropin-releasing hormone receptor and its splice variants.

In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%-65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.

Amino Acid Sequence↗

PubMed1999

Characterization of a brain-specific nuclear LIM domain protein (FHL1B) which is an alternatively spliced variant of FHL1.

We have amplified and sequenced a novel, alternatively spliced variant of a human gene coding for the four-and-a-half LIM domain protein 1 (FHL1). This gene is located at chromosome Xq27 and the spliced variant is named FHL1B. The ORF of FHL1B cDNA codes for a LIM-only protein that possesses a zinc finger and three tandem repeats of LIM domains at the N-terminus with an active bipartite nuclear localization signal (NLS) motif and a possible RBP-J binding region at the C-terminus. FHL1B and FHL1 have the same N-terminal three-and-a-half LIM domains but different C-terminal protein sequences, due to the presence of an additional alternative exon 4b in FHL1B causing a frame-shift in the 3'coding region. RT-PCR results revealed that the expression of FHL1 is not restricted in skeletal muscle and heart, but is widely distributed in other tissues, including brain, placenta, lung, liver, kidney and pancreas, albeit as a low abundance transcript. In contrast, FHL1B is specifically expressed in brain. The C-terminal alternative region in FHL1B is sufficient to localize FHL1B in the nucleus of mammalian cell. FHL1B is probably related to neural differentiation and certain fragile X syndrome.

Alternative Splicing↗

PubMed1991

Isolation of chick tenascin variants and fragments. A C-terminal heparin-binding fragment produced by cleavage of the extra domain from the largest subunit splicing variant.

The extracellular-matrix glycoprotein, tenascin, consists of disulfide-linked subunits of 190, 200 and 230 kDa (the three splicing variants reported in chicken) and usually exists as a six-armed structure under the electron microscope. We used monoclonal antibodies to isolate and characterize different splicing variants and proteolytic fragments obtained from the native protein. Purified monomeric tenascin has a native molecular mass of 216 kDa and is structured as single arms. Tenascin fragments obtained by pepsin digestion bind to monoclonal antibody (mAb) TnM1 which is directed against epidermal-growth-factor-like repeats in the N-terminal half of all subunits. These fragments represent the thin proximal part of the tenascin arms and they are still partially linked to dimers and trimers via disulfide bridges. Using mAb Tn68, that reacts with a fibronectin-type-III repeat towards the C-terminus, a tenascin fragment, generated by treatment with pronase, can be isolated. Ultrastructurally, this fragment looks like the thicker distal part of the tenascin arms. Only the 230-kDa variant of tenascin gives rise to this distal fragment after cleavage within the alternatively spliced fibronectin-type-III repeats. Native tenascin and all fragments containing the distal part of its arms bind to heparin-agarose, whereas the proximal fragments do not. Oligomeric and monomeric tenascin inhibit fibronectin-mediated fibroblast spreading with comparable efficiency when added to the culture medium, while the proximal fragment has no effect. The distal fragment as well as reduced and alkylated tenascin are active in this assay, but only at higher molar concentrations when compared to the native protein.

Animals↗

PubMed1996

Expression of CD44 splice variants in metastatic and non-metastatic mouse tumour cell lines.

Different splice variants of the CD44 cell-surface molecule have been linked to metastasis formation in several animal and human cancers. We have used metastatic CSML-100 and non-metastatic CSML-0 mouse adenocarcinoma cell lines to determine whether variant CD44 molecules could be implicated in the different behaviour of these cells. Two CD44 splice variants containing exons v7-v10 and v8-v10 were detected in the non-metastatic CSML-0. Two other mouse cell lines, the normal mammary gland NMuMG and the mammary pre-neoplastic CL-S1 were also found to express these exons. A short (hematopoietic) CD44 isoform was expressed in the metastatic CSML-100 and three other mouse mammary tumour cell lines. Overexpression of v7-v10 and v8-v10 CD44 variants in CSML 100 cells did not decrease their metastatic potential.

Alternative Splicing↗

PubMed2003

Differential modulation of Kv4 kinetics by KCHIP1 splice variants.

The beta-subunits of the KChIP family modulate properties and expression level of Kv4 channels. We report the cloning of the first splice variant of KChIP1 (KChIP1b) which contains an extra exon, rich in aromatic residues, in the amino terminus. Both splice variants interacted equally well with Kv4.2 subunits based on confocal imaging and upregulation of current density (more than five-fold). No effects on the voltage dependence of activation or inactivation were noted. However, the effects on the kinetics of recovery from inactivation were opposite: KChIP1b induced a slow component in the recovery (tau approximately 1.2 s), in contrast to the increased recovery rate (tau = 125 ms) with KChIP1a. Accordingly, frequency-dependent accumulation of inactivation was enhanced by KChIP1b but reduced by KChIP1a. Since Kv4.2 channels are involved in protection against back propagating action potentials in dendritic spines, a differential expression of either splice variant could shape the dendritic function.

Alternative Splicing↗

PubMed2004

Cloning and identification of NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A.

AIM: To clone, identify and study new NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A. METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR) was conducted to amplify NS5ATP2 gene. RESULTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully. CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.

Antigens, Polyomavirus Transforming↗

PubMed2001

Human angiotensin II type 1 receptor isoforms encoded by messenger RNA splice variants are functionally distinct.

Human tissues that express the angiotensin II (Ang II) type 1 receptor (hAT(1)R) can synthesize four distinct alternatively spliced hAT(1)R mRNA transcripts. In this study, we show that the relative abundance of these mRNA transcripts varies widely in human tissues, suggesting that each splice variant is functionally distinct. Here we demonstrate, for the first time, that the hAT(1)R-B mRNA splice variant encodes a novel long hAT(1)R isoform in vivo that has significantly diminished affinity for Ang II (i.e. >3-fold) when compared with the short hAT(1)R isoform (encoded by hAT(1)R-A mRNA splice variant). This reduced agonist affinity caused a significant shift to the right in the dose-response curve for Ang II-induced inositol trisphosphate production and Ca(2+) mobilization of the long hAT(1)R when compared with that of the short hAT(1)R. The functional differences between these isoforms allows Ang II responsiveness to be fine-tuned by regulating the relative abundance of the long and short hAT(1)R isoform expressed in a given human tissue.

Adrenal Cortex Neoplasms↗