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PubMed1993

[DNA probes for the alternative splicing region of the 6th exon of the human CSF-1 gene. Polymerase chain reaction and subcloning].

The DNA probes--pA6-CSF-1 and pA2-CSF-1 for the alternative splicing region of the 6 exon human CSF-1 gene were prepared using PCR and subsequent subcloning in pUC19 plasmid at the XmaI/BamHI sites. Due to the insert sequencing and blotting of human leukocytes DNA, the DNA probes obtained can be useful for screening of mutations in the human CSF-1 gene.

Alternative Splicing↗

PubMed2000

Regulation of kinetic properties of GluR2 AMPA receptor channels by alternative splicing.

The four subunits of the AMPA-type glutamate receptor (GluR1-GluR4 or GluR-A-GluR-D) exist in two distinct forms, flip and flop, generated by alternative splicing of a 115 bp region. The GluR2 subunit plays a key role in determining the functional properties of the AMPA receptor channel. In this study, we examined the differences in kinetic properties between the flip and flop splice variants of the GluR2 subunit expressed in Xenopus oocytes using fast agonist application techniques. Glutamate was applied to outside-out patches from oocytes with piezo-driven double-barreled application pipettes. Because homomeric receptor channels composed of the edited form of GluR2 (GluR2R) produce no appreciable current responses, we expressed the unedited form of GluR2 (GluR2Q) in oocytes, which produced large current responses sufficient for analysis of the kinetic properties. The time constant for desensitization during application of 1 mM glutamate was 5.89 +/- 0. 17 msec (n = 50) in flip and 1.18 +/- 0.05 msec (n = 37) in flop. The deactivation time constant was 0.62 +/- 0.06 msec (n = 10) in flip and 0.54 +/- 0.05 msec (n = 10) in flop. The steady-state nondesensitizing current was 6.8 +/- 0.4% (n = 53) of the peak current in flip, whereas it was almost negligible in flop, being only 1.1 +/- 0.1% (n = 36). The slower desensitization kinetics and larger steady-state current responses in the flip variant were also observed in heteromeric receptors assembled from GluR2Q/GluR2R. Thus, desensitization occurred much more prominently in the flop variant in the recombinant GluR2 receptor channels.

Alternative Splicing↗

PubMed1993

Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system.

Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p185erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.

Alternative Splicing↗

PubMed1999

Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell-type specific binding.

We have investigated the impact of cellular environment on the neurite outgrowth promoting properties of the alternatively spliced fibronectin type-III region (fnA-D) of tenascin-C. FnA-D promoted neurite outgrowth in vitro when bound to the surface of BHK cells or cerebral cortical astrocytes, but the absolute increase was greater on astrocytes. In addition, different neurite outgrowth promoting sites were revealed within fnA-D bound to the two cellular substrates. FnA-D also promoted neurite outgrowth as a soluble ligand; however, the actions of soluble fnA-D were not affected by cell type. Therefore, we hypothesized that different mechanisms of cellular binding can alter the growth promoting actions of bound fnA-D. We found that fnA-D utilizes two distinct sequences to bind to the BHK cell surface as opposed to the BHK extracellular matrix. In contrast, only one of these sequences is utilized to bind to the astrocyte matrix as opposed to the astrocyte surface. Furthermore, Scatchard analysis indicated two types of receptors for fnA-D on BHK cells and only one type on astrocytes. These results suggest that active sites for neurite outgrowth within fnA-D are differentially revealed depending on cell-specific fnA-D binding sites. Therefore, the function of tenascin-C and its various domains must be considered in terms of cellular context.

Alternative Splicing↗

PubMed2003

Alternative splicing generates a novel non-secretable resistin isoform in Wistar rats.

Resistin is a secreted adipose tissue hormone that belongs to the resistin-like molecule family. We report here a new alternatively spliced isoform of the rat resistin gene, named S-resistin (short resistin), detected in adipose tissue by reverse transcription-polymerase chain reaction (RT-PCR). A comparison of this cDNA variant and genomic sequences indicates the lack of the second exon containing the secretory consensus signal. Both cDNAs, resistin and S-resistin, were carboxy-tagged with FLAG epitope and transiently expressed in cultured cell lines. While the resistin-FLAG construct gives the expected pattern for a secretion protein, the S-resistin-FLAG construct yielded a predominant nuclear staining. These results indicate that this splicing event regulates the fate and probably the function of the mature protein.

3T3-L1 Cells↗

PubMed1995

Proteolysis of major histocompatibility complex class II-associated invariant chain is regulated by the alternatively spliced gene product, p41.

Invariant chain (Ii) is an intracellular type II transmembrane glycoprotein that is associated with major histocompatibility complex class II molecules during biosynthesis. Ii exists in two alternatively spliced forms, p31 and p41. Both p31 and p41 facilitate folding of class II molecules, promote egress from the endoplasmic reticulum, prevent premature peptide binding, and enhance localization to proteolytic endosomal compartments that are thought to be the sites for Ii degradation, antigen processing, and class II-peptide association. In spite of the dramatic and apparently equivalent effects that p31 and p41 have on class II biosynthesis, the ability of invariant chain to enhance antigen presentation to T cells is mostly restricted to p41. Here we show that degradation of Ii leads to the generation of a 12-kDa amino-terminal fragment that in p41-positive, but not in p31-positive, cells remains associated with class II molecules for an extended time. Interestingly, we find that coexpression of the two isoforms results in a change in the pattern of p31 degradation such that endosomal processing of p31 also leads to extended association of a similar 12-kDa fragment with class II molecules. These data raise the possibility that p41 may have the ability to impart its pattern of proteolytic processing on p31 molecules expressed in the same cells. This would enable a small number of p41 molecules to modify the post-translational transport and/or processing of an entire cohort of class II-Ii complexes in a manner that could account for the unique ability of p41 to enhance antigen presentation.

Alternative Splicing↗

PubMed1995

Alternative splicing of ED-A and ED-B sequences of fibronectin pre-mRNA differs in chondrocytes from different cartilaginous tissues and can be modulated by biological factors.

The alternative splicing of the ED-A and ED-B segments of fibronectin pre-mRNA was examined in epiphyseal, costal, and meniscal cartilage from 3-week-old beagles and in nasal, tracheal, articular, and meniscal cartilage from 1- and 2-year-old Labrador retrievers. In contrast to the 100% expression of ED-B(+) mRNA that has been reported for embryonic chick cartilage (Bennett, V.D., Pallante, K.M., and Adams, S.K. (1991) J. Biol. Chem. 266, 5918-5924), all cartilages studied expressed both the ED-B(+) and ED-B(-) forms of fibronectin mRNA with the exception of the trachea, in which expression was 100% ED-B(-). Of all cartilages studied, only the meniscus had detectable levels of ED-A(+) mRNA. Placing articular cartilage chondrocytes in primary monolayer culture dramatically up-regulated the expression of ED-A(+) mRNA to 25% of the total, and this expression was further increased by the addition of transforming growth factor beta 1 or fucoidan to the culture medium. The expression of ED-B(+) mRNA remained at about 18% in the cultured chondrocytes and was not further affected by either transforming growth factor beta 1 or fucoidan. In contrast, dibutyryl cyclic adenosine monophosphate decreased the relative expression of both the ED-A(+) and ED-B(+) forms of fibronectin pre-mRNA. We concluded that the expression of ED-B(+) fibronectin remains relatively high in chondrocytes from cartilaginous canine tissues (15-35%) with the exception of the trachea, in contrast to the less than 10% expression of ED-B(+) fibronectin reported for other non-fetal tissues.

Alternative Splicing↗

PubMed1998

Ca2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human alpha1C subunit gene.

L-type Ca2+ channels are important targets for drugs, such as dihydropyridines (DHPs), in the treatment of cardiovascular diseases. Channel expression is regulated by alternative splicing. It has been suggested that in the cardiovascular system tissue-specific expression of different L-type Ca2+ channel splice variants may underlie the observed differences in sensitivities to channel block by DHPs. We investigated the sensitivity of Ca2+ channel splice variants derived from the human alpha1C gene to the DHP isradipine. Among seven alpha1C channels we observed up to 10-fold differences in IC50 values for isradipine, as well as changes in the voltage dependence of DHP action.

Alternative Splicing↗

PubMed2002

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.

Alternative Splicing↗

PubMed1993

Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock.

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default.

Alternative Splicing↗

PubMed2000

Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability.

We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.

Alternative Splicing↗

PubMed2002

Evolutionarily conserved, DMRT1, encodes alternatively spliced transcripts and shows dimorphic expression during gonadal differentiation in the lizard, Calotes versicolor.

An orthologue of Dmrt1 has been cloned and characterized in the lizard, Calotes versicolor (CvDmrt1). CvDmrt1 encodes alternatively spliced transcripts in genital ridge during gonadal differentiation and in adult testis. Its expression in genital ridge initiates from day 3 and is restricted to mesenchymal cells, which differentiate into the Sertoli cells. Lack of expression in the coelomic epithelium of GR shows that CvDmrt1 expression occurs only in the testicular pathway, and that the Sertoli and granulosa cells in GR may originate from different primordia. From day 25 onwards, the expression shifts majorly towards the germ cells both in testis and ovary. Thus its role in sexual differentiation of C. versicolor, which lacks CSD and TSD, is well documented.

Alternative Splicing↗

PubMed2002

Evolutionarily conserved, DMRT1, encodes alternatively spliced transcripts and shows dimorphic expression during gonadal differentiation in the lizard, Calotes versicolor.

Alternative Splicing↗

PubMed1998

Search for mutations near the alternatively spliced 8-amino-acid exon in the GABAA receptor gamma 2 subunit gene and lack of allelic association with alcoholism among four aboriginal groups and Han Chinese in Taiwan.

The alternatively spliced 8-amino-acid exon for the GABAA receptor gamma2 subunit gene (GABRC2) has been postulated to mediate behavioral actions of alcohol. A rapid search for splice-junction mutations near the 8-amino-acid exon using restriction enzymes which normally recognize sequences near or in the exon gave negative results among 217 alcoholics in four aboriginal groups (Ami, Atayal, Bunun and Paiwan) and Han Chinese in Taiwan. The role of the GABRC2 gene in alcoholism was further assessed by a comparison of allelic frequencies revealed by a NciI RFLP between case and control groups. No significant association of alcohol dependence with GABRC2 alleles was observed. These results suggest that the GABRC2 gene probably does not play an essential role in predisposition to alcoholism in the sample population.

Alcoholism↗

PubMed1995

Higher expression levels of alternatively spliced pX mRNA in human T lymphotropic virus type I asymptomatic carriers positive for antibodies to p40tax protein.

cDNA of human T lymphotropic virus type I (HTLV-I) pX gene mRNA expressed in peripheral blood lymphocytes of asymptomatic carriers was sequenced. One cDNA clone contained a novel splicing acceptor site, indicating an unidentified form of pX mRNA: pX delta 17 delta 37. All 21 asymptomatic carriers expressed some level of alternatively spliced pX mRNA (pX, pX delta 17, p21rex, orfII, or pX delta 17 delta 37). pX and pX delta 17 were the dominant mRNA species among the five pX mRNAs. All pX mRNAs but orfII correlated significantly with amounts of provirus DNA (P < .05). Levels of provirus DNA and pX mRNAs were significantly higher in anti-p40tax-positive carriers than in negative ones. These observations suggest that the pX mRNAs are expressed ubiquitously, with a complex pattern of splicing, and that the presence of anti-p40tax may serve as a marker for a higher virus load and viral replication levels in asymptomatic HTLV-I carriers.

Aged↗

PubMed2000

Alternative splicing for the alpha1 subunit of soluble guanylate cyclase.

Soluble guanylate cyclase (sGC), the receptor for nitric oxide, is a heterodimer consisting of alpha and beta subunits. We investigated the mRNA species for the alpha(1) subunit in human brain, heart, artery and immortalized B-lymphocytes. Three mRNA species were identified in these tissues. The major mRNA species contained the full expression sequence of the alpha(1) subunit. Two other types of mRNA were detected in which 5' sequences were deleted by splicing (506-590 and 412-590). Each of these deletions included the predicted translation start site, indicating that translation of these two alternatively spliced RNA species does not result in the production of full-length alpha(1) subunits. The relative amounts of the two mRNA species with deletions of the translation start site differed significantly between cell lines of immortalized B-lymphocytes from different individuals. sGC enzymic activity was significantly decreased in cellular extracts from cell lines with high proportions of mRNA species containing the deletion 506-590 when compared with extracts from cell lines that contained mostly mRNA without this deletion.

Adult↗

PubMed1993

The tissue specific SmN protein does not influence the alternative splicing of endogenous N-Cam and C-SRC RNAs in transfected 3T3 cells.

The SmN protein is closely related to the constitutively expressed RNA splicing protein SmB but is expressed only in brain and heart tissue. The inclusion of the VASE exon in the N-Cam mRNA and of the N1 exon in the c-src mRNA correlates with the expression pattern of SmN, being observed in brain and heart but not in other tissues and increasing in amount as SmN levels increase during brain development. However, the artificial expression of SmN in cells in which it is normally absent does not affect the pattern of N-Cam and c-src splicing whilst a cell line lacking detectable SmN is able to include the VASE exon. Hence SmN does not appear to be necessary or sufficient for these tissue-specific and developmentally regulated alternative splicing events.

3T3 Cells↗

PubMed1995

Alternative splicing of the dopamine D2 receptor directs specificity of coupling to G-proteins.

Two isoforms of the dopamine D2 receptor have been characterized, D2L (long) and D2S (short), generated by alternative splicing from the same gene. They differ by an in-frame insert of 29 amino acids specific to D2L within the putative third intracytoplasmic loop of the receptor. We have previously demonstrated (Montmayeur, J.-P., Guiramand, J., and Borelli, E. (1993) Mol. Endocrinol. 7, 161-170) that D2S and D2L, although presenting very similar pharmacological profiles, couple differently to the alpha-subunit of guanine nucleotide-binding regulatory proteins (G-proteins). In particular, D2L, but not D2S, requires the presence of the alpha-subunit of the inhibitory G-protein (G alpha i2) to elicit greater inhibition of adenylyl cyclase activity. The insert present in D2L must therefore confer the specificity of interaction with G alpha i2. Thus, we introduced substitution mutations within the D2L insert. These mutant receptors were expressed in JEG3 cells, a G alpha i2-deficient cell line, scoring for those presenting an increased inhibition of adenylyl cyclase by dopamine. Our analysis identified two mutants, S259/262A and D249V, with these properties. These results clearly show that the insert present in D2L plays a critical role in the selectivity for the G-proteins interacting with the receptor.

Adenylyl Cyclase Inhibitors↗

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