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PubMedDate not supplied

Detecting changes in the relative expression of KRAS2 splice variants using polymerase colonies.

Because splice variants of a gene with multiple isoforms give rise to proteins with different functions, it seems plausible that changes in the expression levels of the splice variants could be a contributing factor to disease. In fact, recent examples in the literature clearly illustrate that altered expression levels of splice variants may play an important role in disease. Furthermore, these works demonstrate that changes in expression levels could potentially be used to (1) monitor disease progression, (2) diagnose disease, and/or (3) determine disease state. In this work an immobilized form of PCR, known as polony technology, was adapted to quantify the relative expression levels of splice variants. Specifically, the relative expression levels of the two splice variants of the oncogene K-ras, namely, K-RAS2A and K-RAS2B, were determined using polony technology.

Biomarkers, Tumor↗

PubMed1994

Cyclothiazide differentially modulates desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor splice variants.

Agonist responses for flip splice variants of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR-A, -C, and -D are more strongly potentiated by cyclothiazide than are those for the flop forms. Cyclothiazide shows both greater efficacy and higher apparent affinity for potentiation of GluR-Aflip versus GluR-Aflop. Consistent with higher affinity for the flip splice variant, recovery from potentiation by cyclothiazide proceeds 30 times more slowly for GluR-Aflip than for GluR-Aflop. In the presence of 300 microM cyclothiazide a 6-fold leftward shift in the kainate dose-response curve for GluR-Aflip but not GluR-Aflop additionally contributes to a difference in potentiation for these splice variants. Although control responses to glutamate show strong desensitization for both splice variants of GluR-A, in the presence of 100 microM cyclothiazide desensitization is strongly attenuated for GluR-Aflip, whereas for GluR-Aflop desensitization remains pronounced but with a rate of onset slowed 50-fold, compared with control. In heteromeric AMPA receptors formed from GluR-A and GluR-B, the flip splice variants are dominant for controlling both recovery from potentiation of responses to kainate and block of desensitization of responses to glutamate. Our results suggest that the flip/flop module could directly contribute to the binding site for cyclothiazide, raising the possibility that this site is located in an extracellular receptor domain.

Animals↗

PubMed2005

MDM2 splice variants and their therapeutic implications.

MDM2 splice variants have now been identified in many different tumor types, and their expression has been associated with advanced disease. However, published data concerning their function is contradictory, and therefore their role in tumorigenesis and their potential as a therapeutic target are unclear. Expression of a specific splice variant, MDM2-B, in a transgenic mouse model results in tumor development; and expression of several splice variants has been shown to enhance tumor formation in Emu-myc transgenic mice. However, expression of similar variants in vitro results in growth inhibition, an observation inconsistent with a transformed phenotype. The observed growth inhibition is p53-dependent, resulting from the binding of splice variants with an intact C-terminal RING finger domain to full-length MDM2 protein. In doing so, p53 can no longer bind MDM2, and p53 activity is elevated. Subsequent inactivation of p53 or p53-mediated apoptosis could contribute to the MDM2 splice variant-mediated tumorigenesis observed in vivo. However, MDM2 splice variants, like full-length MDM2, probably display p53-independent activities. Therefore, the potential for MDM2 splice variants as therapeutic targets will be dependent upon their phenotype within specific tumor types.

Animals↗

PubMed1998

Two new p73 splice variants, gamma and delta, with different transcriptional activity.

p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, alpha, or a shorter beta mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, gamma (splicing out exon 11) and delta (splicing out exons 11, 12, and 13). Both gamma and delta p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73gamma nor p73delta interact with p53, whereas p73gamma showed strong interactions with all p73 isoforms, and p73delta binds efficiently p73alpha and p73gamma but only weakly p73beta. At the functional level, p73gamma is significantly less efficient in activating transcription of the p21(Waf1/Cip1) promoter than p53 or p73beta, whereas the effect of p73delta is intermediate and comparable to that of p73alpha. The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21(Waf1/Cip1) promoter: p73beta is the most efficient in inhibiting colony formation, whereas p73gamma is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.

Alternative Splicing↗

PubMed2006

Identification of novel splice variants of Adhesion G protein-coupled receptors.

Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.

Alternative Splicing↗

PubMed2002

Distinct and opposite diversifying activities of terminal transferase splice variants.

The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3'-->5' exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity.

Alternative Splicing↗

PubMed2003

Identification and characterization of a new human type 9 cGMP-specific phosphodiesterase splice variant (PDE9A5). Differential tissue distribution and subcellular localization of PDE9A variants.

Previously, four splice variants of human cGMP-specific phosphodiesterase (PDE) 9A (PDEs 9A1, 9A2, 9A3 and 9A4) have been identified. In this study, we have cloned a cDNA representing a new human PDE9A variant (PDE9A5). PDE9A5 encodes a protein of 492 amino acids, smaller than PDEs 9A1 and 9A2 but larger than PDEs 9A3 and 9A4. The exon structure of PDE9A5 is different from those of PDEs 9A1, 9A2, 9A3 and 9A4 in that, of the 20 exons of PDE9A gene, it lacks exons 2 and 5. PDE9A5 has been characterized in comparison with PDE9A1, the longest PDE9A variant. PDEs 9A5 and 9A1 have similar enzymatic properties. They both have a high affinity for cGMP with similar Km values (0.39 and 0.25 microM, respectively), although they have slightly different Vmax values (2.55 and 0.96 micromol/min/mg, respectively). They exhibit very similar divalent metal ion dependency and inhibitor sensitivity. Real-time quantitative PCR analysis shows that PDEs 9A5 and 9A1 exhibit differential tissue distribution. They are highly expressed in immune tissues (spleen, lymph node and thymus) and are more abundant in T cells than in B cells, neutrophils and monocytes. When transiently expressed in HEK293 cells, PDEs 9A5 and 9A1 proteins exhibit differential subcellular localization. PDE9A5 localizes exclusively in the cytoplasm, whereas PDE9A1 localizes in the nucleus only. The nuclear localization of PDE9A1 is dependent on a unique pat7 motif. By Western blot analysis, native PDE9A1 is detectable in the nucleus but not in the cytoplasm of T cells. Thus, to our knowledge, PDE9A1 is the only PDE isoform found to localize exclusively in the nucleus. We speculate that the physiological role of the PDE9A diversity may be imparting cGMP-metabolizing ability to specific cellular compartments in appropriate tissues.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Cellular activity of dopamine DA2 receptor splice variants and mutants.

Two splice variants of the dopamine D2 (DA2) receptors-a long (DA2l) and short (DA2s) form-and two corresponding mutants (serine at position 311 replaced by a cysteine) have been described. Using CHO-cells transfected with the genes for the splice variants and their respective mutants and a bioassay based on the online registration of the extracellular acidification rate (ECAR) of intact cells, we investigated the cellular activity upon stimulation of the receptor. We first confirmed that the acute response upon short agonist stimulation was significantly higher for DA2s than for DA2l. However, in contrast to the ongoing opinion, we found that the desensitisation pattern upon long-term agonist treatment was indistinguishable for both wild-type receptors. As far as the corresponding ser311cys mutated receptors are concerned, the concentration-response curves and the desensitisation pattern were superimposable to the corresponding wild-type receptors. Inhibition of protein kinase C (PKC) had very little effect both on the concentration-response curves and on the desensitisation pattern. We conclude that signal transduction following stimulation of DA2 receptors in the CHO expression system is more effective for DA2s than for DA2l. The point mutation in position 311 has little impact on the downstream signalling of DA2 receptors.

Animals↗

PubMed1994

Insulin-inducible changes in insulin receptor mRNA splice variants.

Alternative splicing of insulin receptor pre-mRNA has been shown to be regulated in a tissue-specific and developmental manner. We investigated whether the receptor ligand might regulate the relative distribution of alternatively spliced mRNA in insulin-sensitive cells and found that changes in the relative expression of the two alternatively spliced insulin receptor RNA isotypes expressed in hepatocytes are regulated by insulin. In addition, we observed significant differences (p < or = 0.001) in insulin receptor isotype expression in subjects who were hyperinsulinemic and insulin-resistant versus subjects who were insulin-sensitive. These results support a role for insulin in the regulation of the relative expression of alternatively spliced mRNA expressed in insulin-responsive cells and tissues.

Alternative Splicing↗

PubMed2000

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Amino Acid Sequence↗

PubMed2001

Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling.

Recently we characterized three distinct GnRH receptors in the bullfrog (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we further investigated the expression and function of splice variants, generated from the primary bfGnRHR-3 transcript by exon skipping (splice variant 1), intron retention (splice variants 2 and 3), and/or transcriptional slippage (splice variant 4), apart from the constitutively spliced form (wild-type). Cellular expression and function of the splice variants were examined using a transient expression system. Immunoblot analysis revealed that the wild-type receptor and all splice variant proteins were expressed in transfected HeLa cells with no significant differences in expression levels. These splice variants showed a very low binding affinity to ligand and did not induce signal transduction in response to GnRH treatment. Interestingly, cotransfection of the wild-type with splice variants 2--4, but not with splice variant 1, significantly inhibited wild-type receptor-mediated signaling. Subcellular localization analysis of green fluorescent protein-tagged wild-type and splice variant proteins revealed that the wild-type receptor protein was mainly localized in the cell membrane, whereas the splice variant 1 protein was exclusively detected in the cytoplasm. The splice variant 2--4 proteins, however, were found in both the cell membrane and cytoplasm. The inhibition of wild-type receptor signaling by splice variants 2--4 and the subcellular localization of splice variants 2-4 suggest a possible physical interaction of splice variants 2--4 with the wild-type receptor protein. In addition, the ratio of mRNA levels of the wild-type to splice variants 2--4 significantly varied from hibernation (wild-type < splice variants 2--4) to the prebreeding season (wild-type > splice variants 2--4). Collectively, these results suggest that alternative splicing of the bfGnRHR-3 primary transcript plays a role in fine-tuning GnRH receptor function in amphibians.

Alternative Splicing↗

PubMed1997

Localization and functional expression of splice variants of the P2X2 receptor.

cDNAs encoding three splice variants of the P2X2 receptor were isolated from rat cerebellum. The first variant has a serine/proline-rich segment deleted from the intracellularly located carboxyl-terminal domain of the P2X2 subunit. The second and third variants have the splice site in the second half of the predicted first transmembrane domain. Either a 12-amino acid insertion or a six-amino acid deletion occurs at this position. cRNAs for these isoforms of the P2X2 subunit were injected into Xenopus laevis oocytes and tested for function. ATP evoked inward currents only with the splice variant [designated P2X2(b)] having the 69-amino acid deletion. The potencies of various agonists at the homomeric P2X2(b) receptor were not significantly different from those at the P2X2(a) homomeric channel. However, the P2X2(b) receptor showed significantly lower antagonist sensitivity. In contrast to the nondesensitizing P2X2(a) receptor, prolonged application of ATP produced a more rapid desensitization of the P2X2(b) receptor. When the P2X2(a) and P2X2(b) receptor responses were recorded in transfected mammalian cells, this difference was again found. The change in desensitization may be determined by proline/serine-rich segments and/or phosphorylation motifs that are removed from the tail region in formation of the P2X2(b) subunit. In situ hybridization of the three newly isolated isoforms of the P2X2 subunit was performed at the macroscopic and cellular levels; transcripts for two of them [P2X2(b) and p2x2(c)] but not the third [p2x2(d)], which carries the 12-amino acid addition, were present in many structures in the neonatal rat brain and on sensory and sympathetic ganglia. mRNA for the p2x2(d) splice variant was present only in the nodose ganglion, at a low level.

Adenosine Triphosphate↗

PubMed2004

Functional characterization of Ih-channel splice variants from Apis mellifera.

We isolated splice variants of the AMIH cDNA by means of polymerase chain reaction and homology screening. Splicing at one site generates at least four different channel transcripts (AMIH, AMIHL, AMIHM and AMIHT), which code for ion-channel proteins that vary in the interloop regions between the membrane-spanning domains S4 and S5. HEK293 cells in which the AMIHL splice variants were functionally expressed generated currents that were activated by hyperpolarizing voltage steps. Compared to AMIH, AMIHL cells showed pronounced differences in the voltage dependency of activation: the incorporation of 32 extra amino acids between S4 and S5 shifts the activation curve by +25 mV. Intracellular cAMP made the current-activation potential still less negative and accelerated the activation more effectively than it does in AMIH cells. In vertebrates, functional diversity of Ih-channels is generated by four different genes. In Apis mellifera, splice variants coded by the single gene AMIH could generate a similar diversity.

Alternative Splicing↗

PubMed2026

[Genetic and functional characterization of a novel KIT splicing variant in a Chinese three-generation pedigree with piebaldism].

OBJECTIVES: To investigate the genetic etiology of a three-generation pedigree affected with piebaldism. METHODS: Next-generation sequencing and Sanger sequencing were employed to detect and verify gene variants. Bioinformatics tools were used to predict the effects of candidate variants on splicing and protein function. RT-PCR and Sanger sequencing were further performed to validate the impact of the variant on RNA splicing, and homology modeling was applied to predict its effect on the three-dimensional structure of the KIT protein. The pathogenicity of the variant was then classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the UK Association for Clinical Genomic Science (ACGS). RESULTS: A heterozygous insertion variant near the splice site, c.1990+8_1990+9insTGCACCATTGGAGGTAAA, was identified in the KIT gene in the proband and was found to co-segregate with the phenotype within the family. RT-PCR and cDNA sequencing revealed that this variant led to aberrant splicing during transcription, resulting in a 21 bp in-frame insertion in the mRNA, which encodes an extra 7 amino acids within the tyrosine kinase domain and may thus affect protein function. In silico predictions, together with the experimental findings, supported classification of this variant as likely pathogenic according to relevant variant interpretation guidelines. CONCLUSIONS: The heterozygous splice-site insertion variant KIT:c.1990+8_1990+9insTGCACCATTGGAGGTAAA is the genetic cause of piebaldism in this pedigree.

Genetics diagnosis↗

PubMed1998

Coordinated expression of splice variants for luteinizing hormone receptor messenger RNA during the development of bovine corpora lutea.

We characterized splice variants for LH receptor mRNA in the bovine corpus luteum and examined the levels of expression of all the splice variants during the development of corpus luteum. Total RNA was extracted from bovine corpora lutea, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify a part of the extracellular and transmembrane domains. As a result, four distinct bands were observed on analyses with polyacrylamide gel electrophoresis, and sequencing of the products revealed that the largest form (519 bp) was the full-length A form and that the three shorter forms (438, 253, and 172 bp) were its splicing variants. Splicing patterns of the 438-, 253-, and 172-bp forms are identical to the F, B, and G forms, respectively, found in the ovine ovary. In order to examine the level of expression of all these splice variants simultaneously, semiquantitative RT-PCR was set up and performed. The relative intensities for all four forms of LH receptor mRNA significantly increased (P < 0.05) from Stage I (Days 1-4; Day 1 = day of ovulation) to IIS (Days 5-10, CL weight < 4 g), and from Stage IIS to IIL (Days 5-10, CL weight > or = 4 g). The intensities of all forms for LH receptor mRNA decreased slightly, but not significantly, from Stage IIL to III (Days 11-17). The ratios of the levels for all the forms did not change significantly during the development of the corpus luteum. Determination of the steady-state levels of LH receptor mRNA by Northern blotting showed similar changes during the development of the corpus luteum to changes of the splice variants examined by the semiquantitative RT-PCR. These results suggest that at least three splicing variants of LH receptor mRNA (i.e., F, B, and G forms) exist in the bovine corpus luteum and that the levels of all the forms of LH receptor mRNA increased in a coordinated manner during the development of bovine corpus luteum.

Alternative Splicing↗

PubMed2002

Alternative splice variants of doublecortin-like kinase are differentially expressed and have different kinase activities.

Alternative splicing of mRNA transcripts expands the range of protein products from a single gene locus. Several splice variants of DCLK (doublecortin-like kinase) have previously been reported. Here, we report the genomic organization underlying the splice variants of DCLK and examine the expression profile of two splice variants affecting the kinase domain of DCLK and CPG16 (candidate plasticity gene 16), one containing an Arg-rich domain and the other affecting the C terminus of the protein. These splice alternatives were differentially expressed in embryonic and adult brain. Both splice variants disrupted DCLK PEST domains; however, all splice variants remained sensitive to proteolysis by calpain. The adult-specific C-terminal splice variant of DCLK had reduced autophosphorylation activity, but similar kinase activity for myelin basic protein relative to the embryonic splice variant. The splice variant adding an Arg-rich domain gained an autophosphorylation site at Ser-382. Although this protein isoform was expressed mainly in the adult brain, the phosphorylated form was strongly enriched in embryonic brain and adult olfactory bulb, suggesting a possible role in migrating neurons.

Alternative Splicing↗

PubMed2004

A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase beta (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40-60%), whereas the frequency of HPRT splice variants is considerably lower (approximately 1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed.

Alternative Splicing↗

PubMed2001

Predicting splice variant from DNA chip expression data.

Alternative splicing of premessenger RNA is an important layer of regulation in eukaryotic gene expression. Splice variation of a large number of genes has been implicated in various cell growth and differentiation processes. To measure tissue-specific splicing of genes on a large scale, we collected gene expression data from 11 rat tissues using a high-density oligonucleotide array representing 1600 rat genes. Expression of each gene on the chip is measured by 20 pairs of independent oligonucleotide probes. Two algorithms have been developed to normalize and compare the chip hybridization signals among different tissues at individual oligonucleotide probe level. Oligonucleotide probes (the perfect match [PM] probe of each probe pair), detecting potential tissue-specific splice variants, were identified by the algorithms. The identified candidate splice variants have been compared to the alternatively spliced transcripts predicted by an EST clustering program. In addition, 50% of the top candidates predicted by the algorithms were confirmed by RT-PCR experiment. The study indicates that oligonucleotide probe-based DNA chip assays provide a powerful approach to detect splice variants at genome scale.

Algorithms↗