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PubMed2006

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins.

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.

Adaptor Proteins, Signal Transducing↗

PubMed1995

The role of CD44 splice variants in human metastatic cancer.

The large family of CD44 splice variants are likely to serve multiple functions in the embryo and in the adult organism. This is reflected in their complex patterns of expression. In molecular terms these functions are largely unknown. Certain splice variants (CD44v) can promote the metastatic behaviour of cancer cells. In human colon and breast cancer the presence of epitopes encoded by exon v6 on primary resected tumour material indicates poor prognosis. Metastasis-promoting splice variants differ from those that seem not to have a role in the induction of metastasis by the formation of homomultimeric complexes in the plasma membrane of cells. This may increase their affinity to ligands such as hyaluronate. The affinity can be further regulated over a range from low to very high by cell-specific modification. The fact that CD44v epitopes are found on normal epithelial cells such as skin, cervical epithelium and bladder enforces cautious evaluation of the significance of CD44v expression in human cancer. Nevertheless, certain epitopes can serve as tools in early diagnosis of certain cancers and will facilitate the development of specific targeted therapy.

Biomarkers, Tumor↗

PubMed2005

Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue.

Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Expression of EDA+ and EDB+ fibronectin splice variants in bone.

The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT-PCR and LightCycler-based real-time quantitative RT-PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT-PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low-turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones.

Adolescent↗

PubMed2005

[Prognostic significance of survivin splicing variants in expression gastric cancer].

OBJECTIVE: To study the expression and associations of three survivin splicing variants in gastric cancer and normal gastric mucosa, and to evaluate the prognostic significance. METHODS: Real time quantitative RT-PCR was used to detect the expression of three survivin splicing variants in tumor and matched normal gastric mucosa specimens from 77 cases with gastric cancer. RESULTS: The expression of three survivin splicing variants than upregulated significantly in gastric cancer than those in normal mucosas (P< 0.01). In cancer tissues, the expression rates of survivin, survivin-2B, survivin-deltaEx3 were 100%, 79.8% (61/77), 64.9% (50/77) respectively. The survival rate was significantly lower in the patients with high survivin expression than those with low survivin expression (P< 0.01). CONCLUSION: Among three survivin splicing variants, the expression level of wild-type survivin mRNA is an important predictor for prognosis.

Adult↗

PubMed1995

Splice variants of CD44 in human cervical cancer stage IB to IIB.

Aberrant expression of the cell adhesion molecule CD44 has been detected in human tumors and the expression of specific CD44 isoforms (splice variants) has been shown to be associated with metastasis and poor prognosis in human malignancies. We used three different variant exon sequence-specific murine monoclonal antibodies to epitopes encoded by exon v5, exon v6, or exon v7-v8 of human variant CD44 to study the expression of CD44 splice variants by immunohistochemistry in human cervical cancer. One-hundred five patients with surgically treated squamous cell carcinomas of the cervix stages IB to IIB were included in the study. CD44 splice variants CD44v5, CD44v6, and CD44v7-8 were detected in 70, 67, and 26%, respectively. Tumors expressing exon v6 had significantly more often metastasized to the pelvic nodes (58 vs 79%, P = 0.04). Expression of exon v6 was significantly correlated with a greater probability of vascular space invasion (73 vs 50%, P = 0.04) and a significantly lower rate of inflammatory stromal reaction (48 vs 78%, P = 0.004). Patients suffering from tumors expressing splice variant CD44v6 showed poorer overall survival (P = 0.03). In cases with negative pelvic lymph nodes we found a poorer prognosis when tumors expressed CD44v6 (P = 0.01) or CD44v7-8 (P = 0.02). Among the investigated CD44 splice variants expression of exon v6 is the most promising prognostic marker in surgically treated cervical cancer.

Adult↗

PubMed1997

Expression of tenascin-C by human endometrial adenocarcinoma and stroma cells: heterogeneity of splice variants and induction by TGF-beta.

Localization of tenascin-C in vivo and cell culture experiments in vitro have provided evidence for stromal production of tenascin-C in malignant tumors of a variety of organs. Here we raised the question of whether the mesenchymal stroma in the case of endometrial adenocarcinoma is the unique source of tenascin-C. Therefore, the expression of tenascin-C mRNA by human endometrial adenocarcinoma cells and endometrial stroma cells was investigated. Several preparations of endometrial stroma cells produced tenascin-C mRNA. Using a serum-free defined cell culture medium, production of tenascin-C mRNA could be increased by adding either serum or 20 ng TGF-beta/mL to the cell culture medium. Reverse transcriptase polymerase chain reaction analysis revealed that five out of six endometrial adenocarcinoma cell lines produced tenascin-C mRNA. Northern blot experiments and ribonuclease protection assays provided evidence that the number of copies of tenascin-C mRNA was small. Analysis of expressed splice variants by reverse transcriptase polymerase chain reaction analysis revealed the abundance of one major splice variant that lacked all potential alternatively spliced fibronectin type-III-like repeats. Regarding larger splice variants, all fragment sizes that could theoretically originate from seven alternatively spliced fibronectin type-III-like repeats were observed. Evaluating relative signal intensities, the splice variants containing a single fibronectin type-III-like repeat and the variant possessing all but one alternatively spliced repeats were most frequent. In summary, evidence is provided that tenascin-C can originate from both tissue compartments of the human endometrium stroma and (tumor) epithelium. Splice variant analysis revealed a high number of splice variants and a relative high proportion of variants that have so far been regarded as minor constituents of expressed tenascin-C.

Adenocarcinoma↗

PubMed2002

Expression of MUCI splice variants correlates with invasive growth of breast cancer cell lines.

On the basis of alternative splicing the human breast cancer associated MUCI gene codes for different protein products. MUCI splice variants A and B have been shown to be determined by a single A/G nucleotide polymorphism (SNP) in exon 2 of the MUCI gene. We now describe two new splice variants C and D and show by that in human breast cancer cell lines there is selective co-expression of these variants, namely co-expression of variants A and D. We have found that the expression of variants C and D is also determined by the same SNP in exon 2. Since the overexpression of MUCI proteins has been associated with increased invasive behavior of cancer cell lines, we quantitatively determined the mRNA expression levels of the splice variants A, B, C, and D in breast cancer cell lines and correlated them with the in vitro invasiveness of these cell lines. We revealed a significant correlation between the lack of MUCI splice variants B and C and the invasiveness of the cell lines tested. Furthermore, we showed that concomitant expression of variants A and D is associated with a GG in the genotype. These findings suggest that the invasive behavior of breast cell lines may depend on different expression patterns of the MUCI gene determined by a genetic polymorphism.

Alternative Splicing↗

PubMed2004

Differential distribution of splice variants of estrogen receptor beta in human testicular cells suggests specific functions in spermatogenesis.

A growing number of estrogen receptor beta (ER beta) splice variants are reported. Several of these have been discovered in testis, but with few exceptions little is known about their cellular localization. The aim of this study was to identify and elucidate the mRNA expression pattern of the different ER beta splice variants in human testicular cells. Northern analysis was performed on whole testis and fractions enriched in germ cells from untreated men and from estrogen-treated men undergoing sex change surgery. Probes were constructed in order to systematically screen for and identify various ER beta splice variants. Several ER beta bands were observed in the human testis, in which splice variants constituted the major part of total ER beta transcripts. Interestingly, only two ER beta wild-type transcripts were detected. These seem to be virtually absent from the haploid germ cells and are probably mainly located in somatic cells and/or primary spermatocytes. Several novel ER beta deletion variants were found in high levels in the haploid germ cell fractions and were nearly absent in testicular cells from the estrogen-treated men. The cell-dependent distribution raises the question whether splice variants may have specific functions in spermatogenesis, and whether the differential splicing of ER beta is regulated in a cell-specific manner.

Adolescent↗

PubMed1998

Novel splice variants of the voltage-sensitive sodium channel alpha subunit.

In this study, we have identified novel splice variants of the Na+ channel alpha subunit mRNA from cultured rat astrocytes and neuroblastoma cells. These splice variants are characterized by premature truncation or deletion of a segment in the third domain of the Na+ channel alpha subunit. The expression of three splice variants was upregulated by exposure to 1 mM dibutyryl cAMP in spinal cord astrocytes but not in cerebral astrocytes and in B50 and B104 neuroblastoma cells. The calcium ionophore 1 microM A23187, did not influence the expression of splice variants in either astrocytes or neuroblastoma cells. These findings suggest that spinal cord astrocytes may maintain a unique regulatory pathway that participates in the control of Na+ channel mRNA expression.

Alternative Splicing↗

PubMed1997

Isolation of chicken alpha ENaC splice variants from a cochlear cDNA library.

Three splice variants of the alpha subunit of the amiloride-sensitive epithelial sodium channel (alpha ENaC) have been isolated from a chicken cochlear cDNA library. A PCR product, generated from the cochlear library using degenerate primers to regions of homology between the rat alpha ENaC and the degenerin Mec-4, was used as a probe. The three splice variant cDNAs with sizes of 2321, 3399 and 3845 bp correspond to transcripts of 2.5, 3.5 and 3.9 kb as detected by Northern blot analysis. The 3399 bp clone differs from the 2321 clone solely by the addition of 1079 bases in the 3'-non-coding region. Both these cDNAs code for an identical predicted protein of 637 amino acids which has 68% similarity to the rat alpha ENaC, and is probably the chicken homologue of alpha ENaC. The third cDNA of 3845 bp is similar to the 3399 bp clone but includes two exons within the open reading frame. The first of these exons introduces a premature stop codon resulting in a truncated predicted protein of 434 amino acids. Northern blot analysis shows expression of the 2.5 and 3.5 kb transcripts in cochlea and colon, the 2.5 kb transcript in cartilage, whilst the 3.9 kb transcript is only detected in cochlea. No expression is detected in brain, liver, and heart nor, most notably, in lung or kidney.

Alternative Splicing↗

PubMed2003

Developmental expression and subcellular localization of wallaby gonadotropin-releasing hormone receptor and its splice variants.

The developmental expression of gonadotropin-releasing hormone receptor (GnRH-R) and its splice variants was examined in the gonads of tammar wallaby pouch young in order to elucidate the functional role of GnRH-R in the developing testis and ovary. Wallaby GnRH-R, like eutherian GnRH-Rs, contains three exons and two introns. In the present study, the transcripts of two splice variants (GnRH-R Delta 1 and GnRH-R Delta 2) were cloned from the pituitary. GnRH-R Delta 1 contained a 291 bp deletion from nucleotide positions 232 to 522 within exon 1. This transcript appears to be distinctive in the wallaby and has not been reported in other species. GnRH-R Delta 2 contained a 220 bp deletion from nucleotide positions 523 to 742, corresponding to exon 2. We examined the subcellular localization of the wild type GnRH-R and its splice variants with confocal microscopy, showing that both the wild type receptor and the splice variants were membrane-associated molecules. The different pattern of expression of the wild type receptor and the variants transcripts found in adult and neonatal tissues suggests a specific developmental regulation of the GnRH-R Delta 2 transcript. In addition, the developmental expression of the GnRH-R and GnRH-R Delta 1 transcripts showed a possible association with key physiological events during gonadal development in the wallaby pouch young, suggesting that GnRH-R may be involved in the regulation of early development in the testis and ovary.

Aging↗

PubMed2001

Expression and function of TRAF-3 splice-variant isoforms in human lymphoma cell lines.

TRAF-3 gene products are signaling adaptor molecules required for lymphocytes to mediate T-dependent antibody responses in vivo. Previous work identified 8 splice-variant TRAF-3 mRNA species by RT-PCR that have the potential to encode novel isoforms, seven of which induce NF-kappaB activation when over-expressed in 293 cells. Here, their expression was characterized by RNAse protection assay, which showed the T cell line Jurkat D1.1 and the B cell lines BJAB, Daudi, and Raji each expressed mRNA encoding TRAF-3 splice-variants in approximately the same rank order (from highest to lowest); TRAF-3 Delta103aa, Delta83aa, full-length, Delta25aa, Delta52aa, Delta56aa, Delta27aa, and Delta221aa mRNA. The TRAF-3 Delta130aa mRNA was not detectable in any of the cell lines examined. The functional effect of over-expressing each TRAF-3 splice-variant on NF-kappaB activation was studied in the TRAF-5-responsive B cell line, BJAB. Of the seven TRAF-3 splice-variant isoforms that induce NF-kappaB activation in 293 cells, only TRAF-3 Delta27aa, Delta103aa, or Delta130aa induce NF-kappaB activation in BJAB cells. Together, these data indicate that a number of TRAF-3 splice-variant mRNAs are expressed and function in B and T lymphoma lines, which suggests that certain TRAF-3 splice-variant isoforms may participate in mediating the known functions of the TRAF-3 gene in lymphocytes.

Alternative Splicing↗

PubMed2004

Molecular determinants responsible for differences in desensitization kinetics of AMPA receptor splice variants.

Flip (i) and flop (o) alternatively spliced variants of the four glutamate AMPA receptor subunits (GluR1-4) are differentially expressed in the CNS and can display distinct rates of desensitization that contribute to the heterogeneity of native AMPA receptor-dependent synaptic responses. In the present study, we initially compared the kinetics of desensitization in response to fast application of glutamate (1 mm) for the eight different homomeric recombinant human AMPA receptors (hGluR1-4i and o) heterologously expressed in mammalian cells. Consistent with previous reports on recombinant rat AMPA receptors, the time constants of desensitization between human GluR1i and GluR1o receptors were the same, whereas the flip isoforms for GluR2-4 receptors exhibited significantly slower rates of desensitization compared with the flop isoforms. To identify the molecular determinants responsible for these functional differences, the effects of exchanging amino acid residues in the flip-flop cassette of GluR2i and GluR2o were investigated. Three amino acid residues in the flip-flop region (Thr765, Pro766, and Ser775 in flip and Asn765, Ala766, and Asn775 in flop) were identified that contribute to splice-variant differences in the rate of desensitization. Recent structural data show that these three residues are located on helix J, which forms part of the intradimer interface of AMPA receptor ligand-binding cores, and that the stability of this interface may regulate desensitization. The present results suggest that these three residues may confer differences in flip and flop receptor desensitization rates by directly and/or indirectly influencing the stability of the interface between adjacent subunits.

Alternative Splicing↗

PubMed1995

A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial cancer cells.

Signal transduction of cytokine receptors is mediated by the JAK family of tyrosine kinases. Recently, the kinase partners for the interleukin (IL)-2 receptor have been identified as JAK1 and JAK3. In this study, we report the identification of splice variants that may modulate JAK3 signaling. Three splice variants were isolated from different mRNA sources: breast (B), spleen (S), and activated monocytes (M). Sequence analysis revealed that the splice variants contain identical NH2-terminal regions but diverge at the COOH termini. Analyses of expression of the JAK3 splice isoforms by reverse transcriptase-polymerase chain reaction on a panel of cell lines show splice preferences in different cell lines: the S-form is more commonly seen in hematopoietic lines, whereas the B- and M-forms are detected in cells both of hematopoietic and epithelial origins. Antibodies raised against peptides to the B-form splice variant confirmed that the 125-kDa JAK3B protein product is found abundantly in hematopoietic as well as epithelial cells, including primary breast cancers. The lack of subdomain XI in the tyrosine kinase core of the B-form JAK3 protein suggests that it is a defective kinase. This is supported by the lack of detected autokinase activity of the B-form JAK3. Intriguingly, both the S and B splice isoforms of JAK3 appear to co-immunoprecipitate with the IL-2 receptor from HUT-78 cell lysates. This and the presence of multiple COOH-terminal splice variants coexpressed in the same cells suggest that the JAK3 splice isoforms are functional in JAK3 signaling and may enrich the complexity of the intracellular responses functional in IL-2 or cytokine signaling.

Amino Acid Sequence↗

PubMed2004

Splice variants as cancer biomarkers.

Inherited and acquired changes in pre-mRNA splicing have been documented to play a significant role in human disease development and many cancer-associated genes are regulated by alternative splicing. Loss of fidelity, variation of the splicing process, even controlled switching to specific splicing alternatives may occur during tumor progression and could play a major role in carcinogenesis. Splice variants that are found predominantly in tumors have clear diagnostic value and may provide potential drug targets. Moreover, understanding the process of aberrant splicing and the detailed characterization of the splice variants may prove crucial to our understanding of malignant transformation. This review discusses the basic mechanism of alternative splicing, alternative splicing in cancer-associated genes, tools to identify splice variants, and the development of clinical tests based on alternatively spliced biomarkers.

Alternative Splicing↗

PubMed2002

Isolation and differential tissue distribution of two human cDNAs encoding PDE1 splice variants.

A cDNA selection technique has been used to isolate full-length human cDNAs of the phosphodiesterase 1 (PDE1) calcium calmodulin (CaM)-regulated phosphodiesterase gene family. We isolated cDNAs representing multiple splice variants of PDE1A, 1B and 1C from a variety of tissues. Included among these were two novel splice variants for PDE1A and 1B. The first, PDE1A5, encodes a 519-residue protein, which is different from PDE1A1 by the insertion of 14 residues, a divergent carboxy terminus and also differs from PDE1A3 through a divergent amino terminus. Our second novel splice variant represents the first occurrence of a splice variant of the PDE1B gene. PDE1B2 encodes a 516-residue protein and diverges from PDE1B1 by the replacement of the first 38 residues by an alternative 18, which is predicted to be functionally significant. Using the splice variant sequence differences to perform comparative Northern analysis, we have demonstrated that each variant has a differential tissue distribution.

Alternative Splicing↗

PubMed2005

A novel zinc finger gene ZNF468 with two co-expressional splice variants, ZNF468.1 and ZNF468.2.

A novel human zinc finger protein encoding gene ZNF468 was obtained from a fetal brain cDNA library. By BLAST-N analysis we found two different splice variants. We termed the two splice variants ZNF468.1 and ZNF468.2. By BLAST search against the human genome database, ZNF468 was mapped to 19q13.4. The ZNF468.1 cDNA has four exons, and the ZNF468.2 cDNA has one more, between the third and fourth exon. This extra exon creates a difference between the deduced protein N-termini of the two splice variants. The ZNF468.1 cDNA is 3906 bp in length, encoding a 522a a protein, and ZNF468.2 is 4024 bp, encoding a 469-aa-protein. Both proteins contain 11 C2H2-type zinc finger motifs at their C-termini. The N-terminus of the deduced protein of ZNF468.1 has a well-conserved Krüppel-associated box (KRAB) domain that consists of KRAB boxes A and B, whereas the protein of ZNF468.2 does not have the KRAB domain. Tissue distribution of the ZNF468 gene indicates that the two splice variants are widely expressed in normal human tissues, except in heart and brain, and they are also co-expressional.

Alternative Splicing↗