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PubMed1995

Identification of a splice variant of the rat and human mineralocorticoid receptor genes.

The sequence of a splice variant of the rat mineralocorticoid receptor (MR) gene is presented. A cDNA clone corresponding to rat MR was isolated from a rat brain cDNA library. Sequence analysis of the region corresponding to the DNA binding domain revealed the presence of a 12 base pair (bp) insertion. Analysis of mRNA from several rat tissues suggests that the variant is less abundant than the wild type in most tissues. The insertion variant is also a product of the human MR gene, the identical splice variant was also observed in human white blood cell mRNA. Unlike other splice variants reported for the MR, this variant alters the encoded protein by the addition of four amino acid residues in the DNA binding domain. The altered protein may influence the affinity of the MR for mineralocorticoid or glucocorticoid response elements.

Alternative Splicing↗

PubMed2002

Light response of retinal ON bipolar cells requires a specific splice variant of Galpha(o).

Glutamate released onto retinal ON bipolar neurons binds to a metabotropic receptor to activate a heterotrimeric G-protein (G(o)) that ultimately closes a nonspecific cation channel. Signaling requires the alpha subunit (Galpha(o)), but its effector is unknown. Because Galpha(o) is transcribed into two splice variants (alpha(o1) and alpha(o2)) that differ in the key GTPase domain, the next step in elucidating this pathway was to determine which splice variant carries the signal. Here we show by reverse transcription-PCR and Western blots that retina expresses both splice variants. Furthermore, in situ hybridization and immunostaining on mouse retina deficient in one splice variant or the other show that both alpha(o1) and alpha(o2) are expressed by ON bipolar cells but that alpha(o1) is much more abundant. Finally, electroretinography performed on mice deficient for one splice variant or the other shows that the positive b-wave (response of ON bipolar cells to rod and cone input) requires alpha(o1) but not alpha(o2). Thus, the light response of the ON bipolar cell is probably carried by its strongly expressed splice variant, Galpha(o1).

Alternative Splicing↗

PubMed1994

Expression of CD44 splice variants during lymphocyte activation and tumor progression.

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.

Alternative Splicing↗

PubMed2006

Fibronectin EIIIA splicing variant: a useful contribution to forensic wounding interval estimation.

A fibronectin splicing variant, Fn containing an extra type III domain (EIIIA in rat, ED1 or EDA in human) has recently attracted more attention because of its sensitive response in injury of adult tissue. The characteristic that this form is absent in adult tissue while it is commonly expressed in fetal tissues and injured adult tissue is useful in estimation of injury interval in forensic science. The regulation of fibronectin splicing was studied by immunohistochemistry by using monoclonal antibody to EIIIA on sections from paraffin embedded rat skin sample after contusion. The results indicated that epidermic cells and hair follicle epithelium of rat skin were found positive staining at 6h after injury, and the color became darker with prolonged wounding time. Based on the above result, we invented a type of test paper assayed for EIIIA-Fn splicing variant by using immune colloidal gold technique. After dipping one end of test paper in the supernatant fluid of tissue homogenates of injury skin for few minutes, detecting line could be found. It showed that all experimental injured samples revealed positive staining; the darkness of positive staining was dependent on the injury time, while control normal skin cannot found positive staining. It is concluded that there is a close relationship between the expression of EIIIA-Fn splicing variant and wounding interval, and that the gold-labeled test paper can be useful in distinguishing ante- and post-mortem injury, and in estimation of wounding interval in forensic science.

Alternative Splicing↗

PubMed2001

Folylpoly-gamma-glutamate synthetase gene mRNA splice variants and protein expression in primary human leukemia cells, cell lines, and normal human tissues.

Folypoly-gamma-glutamate synthetase (FPGS) is essential for the cytotoxicity of "classical" antifolates and their efficacy in cancer chemotherapy. The expression of the FPGS gene appears controlled by both tissue/lineage-specific and proliferation-dependent mechanisms. Four alternative exon 1 splice variants of the FPGS gene have been described previously, but their significance in gene regulation has not been determined. Furthermore, alternative splicing of the human FPGS gene in normal or transformed cells in vivo has not been reported. We have examined the mRNA expression of these FPGS splice variants in primary human leukemia cells, cell lines, and normal human hematopoietic progenitors using reverse transcription-PCR. Specific primers were designed to amplify splice variants 1, 1A, 1B, and 1C; and full-length FPGS mRNA was amplified using primers for exons 14 and 15 at the 3' end of the gene. In this study, we demonstrate that all four alternative exon 1 variants are expressed in all primary leukemia cells and cell lines (ALL and AML), as well as in normal human hematopoietic progenitors. No significant differences in mRNA expression were detected in primary cells or cell lines for the four exon 1 splice variants. Normal circulating human lymphocytes (peripheral blood mononuclear cells) also expressed mRNA amplified from full-length FPGS and the four exon 1 splice variants, although no detectable FPGS activity was found. Using Western immunoblotting, we show that FPGS protein is expressed in these peripheral blood mononuclear cells; thus, we propose that posttranslational modification(s) is required for expression of a functional FPGS protein in human lymphohematopoietic cells. In addition, poly(A)(+) RNA from normal human adult and fetal tissues and leukemia cell lines was analyzed by Northern blot methodology. Total FPGS mRNA expression showed tissue-specificity, and higher levels were observed in human fetal tissues compared with adult tissues. The data presented herein demonstrate the existence of these FPGS mRNA splice variants in normal and transformed human hematopoietic cells and indicate that alternative splicing of the 5' end of the human FPGS gene does occur in primary cells in vivo. However, its role in regulating the expression of its mRNA remains to be determined.

Alternative Splicing↗

PubMed2006

The opioid ligand binding of human mu-opioid receptor is modulated by novel splice variants of the receptor.

The pharmacological actions of morphine and morphine-like drugs, such as heroin, mediate primarily through the mu-opioid receptor (MOR). It has been proposed that the functional diversity of MOR may be related to alternative splicing of the MOR gene. Although a number of MOR mRNA splice variants have been reported, their biological function has been controversial. In this study, two novel splice variants of the human MOR gene were discovered. Splice variants 1 and 2 (here called the SV1 and SV2) retain different portions of intron I. In vitro translation of SV1 and SV2 produced proteins with the predicted molecular weights. The splice variant proteins were identical to the wild-type MOR-1 up to the first transmembrane domains, but were different after the first intracellular loop domains. SV1 and SV2 of hMOR were present in human neuroblastoma NMB cells and human whole brain confirmed by RT-PCR. In a receptor binding assay, cells expressing the SV1 and SV2 do not exhibit binding to [(3)H]diprenorphine. The formations of MOR.SV1 and MOR.SV2 heterodimers were demonstrated by co-immunoprecipitation and bioluminescence resonance energy transfer between MOR and splice variants. Co-transfection of MOR-GFP and SV-DsRed gene showed that MOR and SV protein co-localized at the cytoplasmic membrane. In NMB cells expressing human MOR gene, transfection of SV1 or SV2 reduced binding activity of the endogenous MOR. These data support a potential role of SV1 and SV2 proteins as possible biological modulator of human mu-opioid receptor.

Alternative Splicing↗

PubMed2001

Novel MUC1 splice variants are expressed in cervical carcinoma.

OBJECTIVES: The MUC1 antigen can be used to identify epithelial cells from the background of hemopoietic cells. The present investigation describes patterns of overexpression of two novel MUC1 splice variants in human cervical carcinoma cell lines. METHODS: RT-PCR was carried out to determine MUC1 splice variants in the cervical cancer cell lines C-4 II, C-33A, DoTc 2 4510, C-4 I, SiHa, HT3, Hs 636 T (C4-I), and HeLa. RESULTS: The novel MUC1 splice variant D was expressed in all cell lines and the novel MUC1 splice variant C was expressed in all cell lines but C-33A. Variants A and B were expressed in all (variant A) and all but one (variant B) cell line. MUC1/REP was expressed in all cell lines and MUC1/SEC was positive in all but two cell lines (C-33 A, DoTc 2 4510). All but one cell line (C-33A) expressed MUC1/X and MUC1/Y, and two cell lines (C-33 A, DoTc 2 4510) did not express MUC1/Z, respectively. MUC1 variants A, D, and REP could be demonstrated consistently among all eight cervical carcinoma cell lines we have examined. CONCLUSIONS: The present study describes the feasibility of detecting a large number of MUC1 variants, including MUC1 variants C and D which are described for cervical carcinoma cells for the first time. Further studies will examine the presence of MUC1 splice variants' expression in human cervical carcinoma tissue.

Alternative Splicing↗

PubMed2002

Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions.

Three splice variants of the renal Na-K-Cl cotransporter (NKCC2 F, A, and B) are spatially distributed along the thick ascending limb of the mammalian kidney. To test whether NKCC2 splice variants differ in ion transport characteristics we expressed cDNAs encoding rabbit NKCC2 F, A, and B in Xenopus oocytes and determined the ion dependence of bumetanide-sensitive (86)Rb influx. The three splice variants of NKCC2 showed dramatic differences in their kinetic behavior. The medullary variant F exhibited 3-4-fold lower affinity than variants A and B for Na(+) and K(+). Chloride affinities also markedly distinguish the three variants (K(m)F = 111.3, K(m)A = 44.7, and K(m)B = 8.9 mm Cl(-)). Thus, the kinetic properties of the NKCC2 splice variants are consistent with the spatial distribution of the variants along the thick ascending limb as they are involved in reabsorbing Na(+), K(+), and Cl(-) from a progressively diluted fluid in the tubule lumen. Variant B also showed an anomalous inhibition of rubidium influx at high extracellular Na(+) concentrations, possibly important in its highly specialized role in the macula densa. The adaptation of the kinetic characteristics of the NKCC2 variants to the luminal concentrations of substrate represents an excellent example of functional specialization and diversity that can be achieved through alternative mRNA splicing.

Alternative Splicing↗

PubMed1995

Tissue-dependent expression of a novel splice variant of the human oestrogen receptor.

We have isolated a novel splice variant of ER mRNA from normal endometrial tissue using RT/PCR. The variant contains an unusual splice junction formed by splicing sequences within exons 4 and 7 together. The translated protein product would be predicted to lack part of exon 4, all of exons 5 and 6 and, due to a missense alignment at the new splice junction, the remaining sequence from exon 7 would be translated out of frame and terminate at the exon 7/8 splice junction. As a result, the protein would lack most of the hormone binding domain (HBD) and the major oestrogen-dependent transactivating region (AF-2), but still contain the DNA binding domain (DNA-BD) and N-terminal transactivating region (AF-1). In contrast to the exon 5 deleted variant of ER (delta 5), which was expressed in both normal endometrium and liver, this novel variant was present in endometrium but not in liver samples. These results confirm that some ER splice variants are expressed in normal, non-malignant oestrogen responsive tissues. In addition, they demonstrate the tissue specific expression of a novel and interesting splice variant of ER in these normal tissues.

Alternative Splicing↗

PubMed1995

Characterization of RET proto-oncogene 3' splicing variants and polyadenylation sites: a novel C-terminus for RET.

The RET proto-oncogene, which encodes a receptor tyrosine kinase, displays multiple alternative splicing variants. Splicing of sequences 3' of exon 19 to generate several coding and untranslated region (UTR) sequences has been previously reported. We have sequenced the full length RET coding region and characterized the transcripts and 3' UTRs generated by alternative splicing of the RET 3' terminus. These analyses were performed using both RET cDNA cloned from a pheochromocytoma library and reverse transcriptase PCR products generated using RNA from a neuroblastoma cell line (LA-N-2). Three different carboxyl termini were identified. In addition to the nine and 51 terminal amino acid forms already known, we identified a third with 43 terminal amino acids predicted to encode a novel RET protein isoform. A total of 3621 base pairs of DNA 3' of exon 19, which spans the alternatively spliced exons and RET UTRs, was sequenced. Four polyadenylation sites were identified. The observed combinations of polyadenylation sites and 3' coding sequence suggest that RET transcripts with up to 10 different 3' sequences and up to 40 different full length RET transcripts may exist.

Adrenal Gland Neoplasms↗

PubMed2002

Smooth muscle tissues express a major dominant negative splice variant of the type 3 Ca2+ release channel (ryanodine receptor).

It is well known that the type 3 Ca(2+) release channel (ryanodine receptor, RyR3) exhibits strikingly different pharmacological and functional properties depending on the tissues in which it resides. To investigate the molecular basis for this tissue-dependent heterogeneity, we examined the primary structure of RyR3 from various tissues by reverse transcription polymerase chain reaction and DNA sequence analysis. As many as seven alternatively spliced variants of RyR3 were detected. Ribonuclease protection assays revealed that one of these splice variants, RyR3 (AS-8a), which lacks a 29-amino acid fragment (His(4406)-Lys(4434)) encompassing a predicted transmembrane helix, was highly expressed in smooth muscle tissues, but not in skeletal muscle, the heart, or the brain. Although the RyR3 (AS-8a) splice variant did not form a functional Ca(2+) release channel when expressed alone in HEK293 cells, it was able to form functional heteromeric channels with reduced caffeine sensitivity when co-expressed with the wild type RyR3. Interestingly, this RyR3 splice variant was also able to form heteromeric channels with and suppress the activity of the type 2 ryanodine receptor (RyR2). Tissue-specific expression of RyR3 splice variants is therefore likely to account for some of the pharmacological and functional heterogeneities of RyR3. These observations also reveal a novel mechanism by which a splice variant of one RyR isoform (RyR3) can suppress the activity of another RyR isoform (RyR2) via a dominant negative effect.

Alternative Splicing↗

PubMed1997

Immunohistochemical detection of CD44 splice variant expression in premalignant lesions of the cervix and benign cervical epithelium.

The expression of specific CD44 splice variants in malignant tumors has been shown to be associated with metastasis and poor prognosis. In previous studies we have detected aberrant expression of the transmembranous cell adhesion molecule CD44 in cervical cancer. Therefore, we now focus on its role in intraepithelial dysplastic lesions of the cervix. Forty-five cervical intraepithelial neoplasias (CIN), grades I to III, were investigated immunohistochemically for three different splice variants of CD44 (v5, v6, and v7-8). A negative correlation of CD44v5 (Kendall correlation coefficient r = -0.25, P < 0.05) expression with the grade of CIN was found. In normal squamous epithelium of the cervix, the staining reaction of CD44v5 was confined to the basal and the parabasal cell layers. In dysplastic lesions a diffuse and weaker staining pattern of the entire epithelium was observed. Our data suggest a loss of CD44v5 epitope during dysplastic transformation. It seems that expression of CD44 splice variants is not a continuous process in the natural history of cervical cancer, and the pattern of CD44 splice variant expression is changed during carcinogenesis.

Cervix Uteri↗

PubMed2000

Mice lacking the long splice variant of the gamma 2 subunit of the GABA(A) receptor are more sensitive to benzodiazepines.

The gamma 2 subunit is required for benzodiazepine modulation of the GABA(A) receptor. Alternate splicing of precursor GABA(A) gamma 2 mRNA results in two splice variants, a short (gamma 2S) and a long (gamma 2L) variant. We investigated the roles of these splice variants in benzodiazepine pharmacology using mice lacking genes for the gamma 2L splice variant. Sleep time responses to midazolam and zolpidem were 20 and 18% greater, respectively, in null allele mice compared with wild-type mice, while responses to nonbenzodiazepine agents such as etomidate and pentobarbital were unchanged. Although the GABA(A) receptor number was not altered in null allele mice, there was a corresponding increase in affinity of brain membranes for benzodiazepine agonists (midazolam, diazepam, and zolpidem), while affinity for benzodiazepine inverse agonists (beta CCM and Ro15-4513) was decreased. These changes were not observed in inbred mice of the parental strains (C57BL/6J and 129/SvJ) used to create the genetically altered mice, indicating that differences between gamma 2L null allele and wild-type mice were unlikely to be simply due to cosegregation of linked alleles. Absence of the gamma 2L splice variant increases the affinity of receptors for benzodiazepine agonists, and is associated with a modest increase in behavioral sensitivity to benzodiazepine agonists. Lack of the gamma 2L subunits may shift the GABA(A) receptor from an inverse agonist-preferring toward an agonist-preferring configuration.

Alternative Splicing↗

PubMed2000

Functional expression of three P2X(2) receptor splice variants from guinea pig cochlea.

ATP has been suggested to act as a neurotransmitter or a neuromodulator in the cochlea. The responses to ATP in different cell types of the cochlea vary in terms of the rate of desensitization and magnitude, suggesting that there may be different subtypes of P2X receptors distributed in the cochlea. Recently three ionotropic P2X(2) receptor splice variants, P2X(2-1), P2X(2-2), and P2X(2-3,) were isolated and sequenced from a guinea pig cochlear cDNA library. To test the hypothesis that these different splice variants could be expressed as functional homomeric receptors, the three P2X(2) receptor variants were individually and transiently expressed in human embryonic kidney cells (HEK293). The biophysical and pharmacological properties of these receptors were characterized using the whole cell patch-clamp technique. Extracellular application of ATP induced an inward current in HEK293 cells containing each of the three splice variants in a dose-dependent manner indicating the expression of homomeric receptors. Current-voltage (I-V) relationships for the ATP-gated current show that the three subtypes of the P2X(2) receptor had a similar reversal potential and an inward rectification index (I(50 mV)/I(-50 mV)). However, the ATP-induced currents in cells expressing P2X(2-1) and P2X(2-2) variants were large and desensitized rapidly whereas the current in those cells expressing the P2X(2-3) variant was much smaller and desensitized slower. The order of potency to ATP agonists was 2-MeSATP > ATP > alpha,beta -MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on all three variants. Results demonstrate that three P2X(2) splice variants from guinea pig cochlea, P2X(2-1), P2X(2-2), and P2X(2-3), can individually form nonselective cation receptor channels when these subunits are expressed in HEK293 cells. The distinct properties of these P2X(2) receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.

Adenosine Triphosphate↗

PubMed2004

Novel splicing variant of the human orphan nuclear receptor Nurr1 gene.

BACKGROUND: Nurr1 is a member of the nuclear receptor superfamily of transcription factors. The objective of the present study was to identify novel splicing variants of the gene in neuronal and non-neuronal tissues and determine their functions. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to screen for Nurr1 splice variants in the adult human central nervous system (CNS) and in other tissues such as lymphocytes, and liver, muscle, and kidney cells. Functional assays of the variants were performed by measuring Nurr1 response element (NuRE) transcriptional activity in vitro. RESULTS: In this study, the authors identified a novel splicing variant of Nurr1 within exon 5, found in multiple adult human tissues, including lymphocytes, and liver, muscle, and kidney cells, but not in the brain or spinal cord. Sequencing analysis showed the variant has a 75 bp deletion between nucleotides 1402 and 1476. A functional assay of the Nurr1-c splicing variant, performed by measuring NuRE transcriptional activity in vitro, detected a 39% lower level of luciferase (LUC) activity (P < 0.05). CONCLUSION: A novel splicing variant of Nurr1 exists in human non-neuronal tissues and functional assays suggest that the variant may act as an alternate transcription regulator.

Adult↗

PubMed2001

Alternatively spliced variant deleting exons 7 and 8 of the human telomerase reverse transcriptase gene is dominantly expressed in the uterus.

The expression level of the human telomerase reverse transcriptase (hTERT) is a rate-limiting determinant of telomerase activity. Several alternatively spliced variants of hTERT transcript are currently known. We have studied the expression of the splicing variants arising in the transcript encoding the reverse transcriptase domain, and have compared this to the telomerase activity in 27 endometria, 14 myometria and 18 endometrial carcinomas. Telomerase activity and the full-length hTERT transcript were observed in endometrial samples from the proliferative and early secretory phases, but not in those from the late secretory phase. Steady-state expression of the hTERT splicing variant entirely lacking exon 7 and exon 8 was observed in the endometria throughout the menstrual cycle. In the analysed myometria, this type of splicing variant was the most commonly detected, and telomerase activity occurred in only three samples. In both endometria and myometria, the expression of the full-length transcript correlated well with the telomerase activity. In each of the endometrial carcinomas, telomerase activity was detected and the full-length transcript was found together with varying combinations of deletion splicing variants. These results suggest that regulation of splicing in the transcript encoding the hTERT reverse transcriptase domain is associated with telomerase activation in uterine tissues.

Adult↗

PubMed2000

Immunocytochemical distribution of the GABA(B) receptor splice variants GABA(B) R1a and R1b in the rat CNS and dorsal root ganglia.

The anatomical distribution of the GABA(B) receptor (GBR) splice variants GBR1a and 1b in the CNS has not previously been studied. In the present study, distribution of the splice variants was mapped using immunohistochemistry. Polyclonal antibodies against splice variant unique epitopes were raised in rabbits. Affinity purified antibodies were used according to routine immunohistochemical procedures in sections from the rat CNS or dorsal root ganglia (DRG). The staining intensity was high in the cerebral cortex but lower in basal ganglia and the hippocampus. In the cerebellum, there was a marked difference in the distribution of GBR1a- and 1b-like immunoreactivity (LI). GBR1a-LI was preferentially localised in the granule cell layer whilst GBR1b-LI was mostly found in Purkinje cells and in the molecular layer. Cell bodies of the deep cerebellar nuclei stained for the GBR1a antibody while terminals surrounding the cell bodies were strongly labelled with the GBR1b antibody. A similar pre- vs postsynaptic pattern was seen in several nuclei ventral or caudal to the cerebellum (e.g. the cochlear nucleus, the facial nucleus, the spinal cord) but not in regions rostral to the cerebellum. In the spinal cord, strong labelling for both antibodies was seen in the dorsal horn. The GBR1b but not the GBR1a antibody stained tanycytes in the epithelium of the 3rd ventricle and in the central canal at the brain stem level. DRG neurons were positive for both the GBR1a and 1b antibody, but the former stained the cells much more intensely. Satellite cells were labelled with the GBR1b antibody. The most important aspect of these findings is that in some nuclei, GBR1b may mediate inhibition of transmitter release while in the same regions, GBR1a may mediate postsynaptic inhibition. Further, the observations support previous findings that GBR1b is the predominant splice variant in Purkinje cells.

Animals↗

PubMed1998

Use of long RT-PCR to characterize splice variant mRNAs.

Recent advances in long reverse transcription (RT)-PCR technology allow the copying of full-length coding regions of large mRNAs in one step. Using long RT-PCR, one can be certain that a given cDNA is derived from a single mRNA. In what to our knowledge is a novel application, we can isolate and characterize splice variants for any given mRNA in a systematic manner. We optimized long RT-PCR to copy the full-length coding region of human multidrug resistance (MDR1) mRNA or the major vault protein (MVP) mRNA in one step, so that only one full-length PCR product was synthesized in each case. Such stringent conditions are necessary to ensure that smaller than full-length products derived from total cell RNA are true splice variants. Twenty MDR1 double-stranded (ds) cDNAs, isolated from either the full-length or one prominent splice-variant DNA band, visualized on agarose gels, were cloned and sequenced. Two were full-length, wild-type in sequence as expected, and the rest were splice-variant mRNAs. Fourteen of the clones were identical and encoded a prominent splice-variant mRNA that can be detected in two tumor cell lines. This approach is shown to be generally applicable to the systematic analysis of splice-variant mRNAs derived from any gene.

Alternative Splicing↗