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PubMed1994

Isolation and characterization of two alternatively spliced complementary DNAs encoding a Xenopus laevis angiotensin II receptor.

We have isolated two cDNAs of 1.7 and 3.0 kb, produced by alternative splicing, that encode a angiotensin II (AII) receptor from a Xenopus laevis heart cDNA library. The two clones had identical coding regions with each other and were found to belong to the G protein-coupled receptor superfamily like the mammalian type 1 AII receptors (AT1); their amino acid sequence was 68.7% homologous with the human AT1 receptor sequence. However, there was a 1.3 kb insertion at the 3'-untranslated region of the longer clone. The insertion contained 9 repeats of an ATTTA motif, suggesting that the two mRNAs undergo distinct post-transcriptional regulation by virtue of a difference in their stability. Although the Xenopus receptor exhibited distinct specificities for AII receptor antagonists compared with mammalian AII receptors, several common characteristics, including the effect of dithiothreitol and guanosine 5'-O-(3-thiotriphosphate), demonstrated that the cloned receptor is a counterpart of the mammalian AT1 receptor. Moreover, the cloned receptor was expressed most abundantly in the Xenopus heart, which is inconsistent with the tissue distribution of mammalian AII receptors. This indicated that the Xenopus heart, unlike that of mammals, plays a major role in the AII-dependent regulation of blood pressure and extracellular fluid volume.

Alternative Splicing↗
PubMed1999

Retinoic acid-dependent upregulation of mouse folate receptor-alpha expression in embryonic stem cells, and conservation of alternative splicing patterns.

The action of retinoic acid (RA) in normal development and differentiation is mediated by changes in the expression of RA-responsive target genes. We used differential display reverse transcriptase polymerase chain reaction (RT-PCR) to identify RA-responsive genes expressed in embryonic stem (ES) cells and found that murine folate receptor-alpha (FR-alpha) expression is rapidly induced by RA treatment. The observed increase in FR-alpha expression occurs within 3h, is independent of protein synthesis and does not occur when ES cells are differentiated by removal of leukaemia inhibitory factor (LIF), evidence that the response to RA is both direct and specific. Two messenger RNA (mRNA) isoforms of FR-alpha featuring novel sequence in the 5' untranslated region (UTR) were cloned, and both were found to be upregulated by RA. Other splice variants in both the 5' UTR and 3' UTR of FR-alpha mRNA were also identified. There is a striking similarity between these splicing patterns and those reported for human FR-alpha, which also generates multiple isoforms by alternative splicing in the 5' and 3' UTR. The conservation of these splicing patterns in the non-coding regions of the FR-alpha gene between mouse and human suggests that these regions, and in particular the 5' UTR, play an important role in regulating expression of this gene.

3' Untranslated Regions↗
PubMed2003

Triiodothyronine affects the alternative splicing of thyroid hormone receptor alpha mRNA.

The c-erbAalpha gene encodes two thyroid hormone receptors, TRalpha1 and TRalpha2, that arise from alternative splicing of the TRalpha pre-mRNA. TRalpha2 is not able to bind triiodothyronine (T(3)) and acts as a weak antagonist of TRs. It has been suggested that the balance of TRalpha1 to TRalpha2 is important in maintaining homeostasis. Here, we study the effect of thyroid hormone on the splicing of TRalpha under various conditions in HepG2 cells. First, T(3) was added to HepG2 cells that endogenously express TRalpha. This resulted in a decrease in the TRalpha1:TRalpha2 mRNA ratio after the addition of 10(-)(8 )M or 10(-)(7 )M T(3). Then, HepG2 cells were incubated with sera from hypothyroid or hyperthyroid patients. Sera from hyperthyroid patients (n=6) decreased the TRalpha1:TRalpha2 ratio compared with HepG2 cells incubated with sera from euthyroid patients (n=8). Sera from hypothyroid patients (n=6) had no effect on the TRalpha1:TRalpha2 ratio but supplementation with T(3) caused a decrease in the ratio. Finally, we tested sera from patients with nonthyroidal illness (NTI; n=17) which showed no effect on TRalpha splicing when compared with controls. Free thyroxine levels in sera from hypo-, eu-, and hyperthyroid patients, but not that of NTI patients, were negatively correlated (P<0.01) to the TRalpha1:TRalpha2 ratio. We next studied the expression of the splicing factors hnRNP A1 and ASF/SF2 (SF2) in relation to the splicing of the TRalpha gene. In HepG2 cells incubated with NTI sera a negative relationship was found between the ratio of hnRNP A1:SF2 and the TRalpha1:TRalpha2 ratio. A high hnRNP A1:SF2 ratio is associated with the use of the distal 5'-splice site. The splicing direction should then change towards TRalpha2, which is indeed the case. Rev-ErbA, which is partly complementary to TRalpha2 and could therefore interfere in the splicing process, did not relate to the TRalpha1:TRalpha2 ratio. In conclusion, high T(3) levels induce a low TRalpha1:TRalpha2 ratio which could protect the cell from excessive T(3)-induced gene expression. In vivo, this might be a mechanism to keep tIssues relatively euthyroid during high serum T(3) levels.

Alternative Splicing↗
PubMed1990

Alternative splicing of the human alpha 2(VI) collagen gene generates multiple mRNA transcripts which predict three protein variants with distinct carboxyl termini.

We recently reported the isolation and sequencing of two classes of human alpha 2(VI) collagen cDNA clones which share common sequences for the first two-thirds of the molecule but contain a different sequence of either 607 or 887 base pairs at their 3' ends (Chu, M.-L., Pan, T.-C., Conway, D., Kuo, H.-J., Glanville, R. W., Timpl, R., Mann, K., and Deutzmann, R. (1989) EMBO J. 8, 1939-1946). In the present study, we report the sequence of another cDNA clone, which is identical to one class of the previously isolated cDNAs except for a 293-base pair insertion between the common and variable regions. Together, the different classes of cDNAs, referred to as the alpha 2C2, alpha 2C2a, and alpha 2C2a' predict three variant alpha 2 chains of type VI collagen with carboxyl globular domains of 429, 328, and 238 amino acid residues, respectively. In order to explore the mechanisms by which the variations are generated, we isolated and characterized the 3' end of the human alpha 2(VI) collagen gene. The carboxyl globular domain was found to be encoded by six exons which appear to delineate its structural subdomains. The exon/intron arrangement clearly demonstrated that the cDNA variants arose from alternative splicing events by mutually exclusive utilization of the last two exons in conjunction with the selective usage of an internal splice acceptor site in the penultimate exon. The presence of the corresponding mature mRNA transcripts (3.2-3.5 kilobase pairs (kb] in human fibroblasts was shown by Northern blot hybridization, S1 nuclease protection assay, and the polymerase chain reaction. The results indicated that the alpha 2C2 mRNA is the major species, whereas the alpha 2C2a and alpha 2C2a' are the minor forms. Northern blot hybridization also revealed an alpha 2(VI) collagen mRNA of 6.0 kb. This mRNA retained a 2.3-kb intron located between the two alternatively spliced exons and predicted a translational product that is the same as the alpha 2C2a variant.

Amino Acid Sequence↗
PubMed1994

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development.

The establishment of the cardiovascular system represents an early, critical event essential for normal embryonic development. An important component of vascular ontogeny is the differentiation and development of the endothelial and endocardial cell populations. This involves, at least in part, the expression and function of specific cell surface receptors required to mediate cell-cell and cell-matrix adhesion. Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) may well serve such a function. It is a member of the immunoglobulin superfamily expressed by the entire vascular endothelium in the adult. It is capable of mediating adhesion by a heterophilic mechanism requiring glycosaminoglycans, as well as by a homophilic, glycosaminoglycan independent, mechanism. It has been shown to regulate the expression of other adhesion molecules on naive T cells. This report documents by RT-PCR and immunohistochemical analysis the expression of PECAM-1 during early post implantation mouse embryo development. PECAM-1 was expressed by early endothelial precursors first within the yolk sac and subsequently within the embryo itself. Interestingly, embryonic PECAM-1 was expressed as multiple isoforms in which one or more clusters of polypeptides were missing from the cytoplasmic domain. The sequence and location of the deleted polypeptides corresponded to exons found in the human PECAM-1 gene. The alternatively spliced isoforms were capable of mediating cell-cell adhesion when transfected into L-cells. The isoforms differed, however, in their sensitivity to a panel of anti-PECAM-1 monoclonal antibodies. These data suggest that changes in the cytoplasmic domain of PECAM-1 may affect its function during cardiovascular development, and are consistent with our earlier report that systematic truncation of the cytoplasmic domain of human PECAM-1 resulted in changes in its ligand specificity, divalent cation and glycosaminoglycan dependence, as well as its susceptibility to adhesion blocking monoclonal antibodies. This is the first report of naturally occurring alternatively spliced forms of PECAM-1 having possible functional implications.

Amino Acid Sequence↗
PubMed2004

Modulation of agrin function by alternative splicing and Ca2+ binding.

The aggregation of acetylcholine receptors on postsynaptic membranes is a key step in neuromuscular junction development. This process depends on alternatively spliced forms of the proteoglycan agrin with "B-inserts" of 8, 11, or 19 residues in the protein's globular C-terminal domain, G3. Structures of the neural B8 and B11 forms of agrin-G3 were determined by X-ray crystallography. The structure of G3-B0, which lacks inserts, was determined by NMR. The agrin-G3 domain adopts a beta jellyroll fold. The B insert site is flanked by four loops on one edge of the beta sandwich. The loops form a surface that corresponds to a versatile interaction interface in the family of structurally related LNS proteins. NMR and X-ray data indicate that this interaction interface is flexible in agrin-G3 and that flexibility is reduced by Ca(2+) binding. The plasticity of the interaction interface could enable different splice forms of agrin to select between multiple binding partners.

Agrin↗
PubMed1999

An alternatively spliced form of CD79b gene may account for altered B-cell receptor expression in B-chronic lymphocytic leukemia.

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Igalpha/Igbeta (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19(+) cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5(+) and CD5(-) B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5(+) and CD5(-) B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 +/- 0.20 SD in normal donors and 0.44 +/- 0.27 SD in B-CLL (P =.01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT --> TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

Antigens, CD↗
PubMed2003

Alternative splicing to exon 11 of human prolactin receptor gene results in multiple isoforms including a secreted prolactin-binding protein.

Endocrine and autocrine prolactin (PRL) exerts effects on normal breast and breast cancer cells, and high serum PRL is a poor prognostic factor for colorectal cancer. Here we tested the hypothesis that short isoforms of the PRL receptor (PRLR) in human tissue regulate the actions of PRL in cancer. Using 3' RACE we isolated five splice variants of the human PRLR (hPRLR), three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9 respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant-negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant-negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.

Alternative Splicing↗
PubMed2003

Cloning, sequencing and tissue distribution of rat flavin-containing monooxygenase 4: two different forms are produced by tissue-specific alternative splicing.

The nucleotide sequence of rat flavin-containing monooxygenase 4 (FMO4) mRNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and 5'/3' terminal extension. Complete cDNA was amplified, cloned, and sequenced from the mRNA obtained from rat kidney and brain. Two different transcripts (short and long) stemming from the splicing of an internal region of 189 bases pair, corresponding to exon 4 were identified. This alternative splicing seems to be specific of the brain. The long cDNA encodes a protein of 560 amino acids with a predicted molecular mass of 63,395 Da. The short cDNA encodes a protein of 497 amino acids with a predicted molecular mass of 55,871 Da. Both of these encoded sequences contain the NADPH- and FAD-binding sites and a hydrophilic carboxyl terminus. These sequences are 80 and 79% identical to the sequences of human and rabbit FMO4. By Northern blotting and/or RT-PCR, the long-form FMO4 mRNA was detected in the rat kidney, intestine, and liver and the short form particularly in the brain. For the first time, the expression of FMO4 protein was demonstrated. By Western blotting using the two different forms of FMO4 antibodies, a long FMO4 protein was detected in the rat kidney, whereas in the rat brain, only the short form of FMO4 was observed.

Alternative Splicing↗
PubMed1990

Cell-type-specific expression of alternatively spliced human fibronectin IIICS mRNAs.

Fibronectin polypeptide diversity is generated to a large extent by alternative splicing of the fibronectin primary transcript at three sites: two extra domain exons encoding extra structural repeats and a region of nonhomologous sequence termed the type-III connecting segment (IIICS). A novel double primer extension assay was developed to identify and quantify simultaneously each of the five human IIICS mRNA splicing variants. Expression of the five IIICS variants was analyzed in a variety of human normal and tumor cell types as well as in human liver. Differences in IIICS expression patterns were observed among different cell types, among fibroblasts of different tissue origins, and between comparable normal and transformed cells. The most predominant cell-type-specific differences were in the abundance of the one IIICS- mRNA variant relative to the four IIICS+ variants. The percentage of O variant (IIICS-) mRNAs within the total fibronectin mRNA pool varied between 3 and 17% among tumor cells and between 7 and 46% among normal cells. The O variant composed 57% of the fibronectin mRNA in liver tissue, correlating with the previously described increased abundance of IIICS- polypeptide subunits in plasma fibronectin, compared with those in cellular fibronectins. Additional cell-type-specific changes among the expression levels of the four IIICS+ mRNA variants are consistent with a proposed model in which regulation of an alternative selection of a 3'splice site predominates over regulation of the selection of a 5' splice site in generating specific patterns of IIICS mRNA expression.

Base Sequence↗
PubMed2002

Identification of an alternatively spliced isoform of the fyn tyrosine kinase.

Two isoforms of the src-family tyrosine kinase p59fyn have been shown to arise through alternative splicing of exon 7 of the fyn gene. These isoforms have been designated fynT, expressed in hematopoietic cells, and fynB, expressed in the brain. Here, we describe a novel isoform, fynDelta7, in which exon 7 is absent. FynDelta7 mRNA transcripts have been identified in peripheral blood mononuclear cells from all individuals tested thus far and semi-quantitative RT-PCR indicates that this fyn transcript is expressed at near wild-type levels. Transcripts coding for the fynDelta7 isoform can be detected in purified B cells, T cells, NK cells, and monocytes, indicating that it is not lineage specific. We further show that the message isolated is functional using an in vitro expression system and by its expression in COS cells.

Alternative Splicing↗
PubMed2003

Genetic polymorphism and sequence evolution of an alternatively spliced exon of the glial fibrillary acidic protein gene, GFAP.

Isoform GFAPepsilon of the human cytoskeletal protein GFAP carries, as the result of alternative splicing of exon 7a of GFAP, a novel 42-amino-acid-long C-terminal region with binding capacity for the presenilin proteins. Here we show that exon 7a is present in a variety of mammals but absent from GFAP of chicken and fish. Comparison of the mouse and human GFAP exons showed an increased rate of nonsynonymous nucleotide substitutions in exon 7a compared to the other exons. This resulted in 10 nonconservative and 2 conservative amino acid substitutions and suggests that exon 7a has evolved under different functional constraints. Exons 7a of humans and higher primates are 100% identical apart from alanine codon 426, which is conserved in only 9% of the human alleles, while 21 and 70% of the alleles, respectively, have a valine or a threonine codon at that position. Threonine represents a potential phosphorylation site, and positive selection of that effect could explain the high allele frequency.

Alternative Splicing↗
PubMed1995

ZEMa, a member of a novel group of MADS box genes, is alternatively spliced in maize endosperm.

The identification of a number of cis-elements which direct gene expression in maize endosperm, and the characterization of corresponding DNA binding proteins, point to the interaction of different classes of transcription factors in this tissue. To assess whether MADS box genes are also involved in maize endosperm development, cDNA and genomic MADS box clones have been isolated. The three cDNA clones ZEM1, ZEM2 and ZEM3 were cloned from a maize endosperm cDNA library using a probe based on sequences conserved in plant MADS box genes. Further transcripts were cloned by RT-PCR experiments and designated ZEM4 and ZEM5. Analysis of the corresponding genomic clones led to the identification of the ZEM2 MADS box gene family, three members of which were characterized sharing 97% sequence identity in corresponding domains. 100% sequence identities between cDNA and one of the genomic clones, conserved exon-intron boundaries and the demonstration of in vivo splicing in a maize endosperm transient expression system, show that the transcripts ZEM1-5 are derived by alternative splicing of ZEMa, one ZEM2 member. The ZEMa transcripts are present in almost all maize tissues, but specific differentially spliced forms accumulate preferentially in maturing endosperm and leaf. The function of the ZEMa gene is discussed in the light of similarities in the expression pattern with members of the human MEF2/RSRF gene family.

Alternative Splicing↗
PubMed1995

Alternate splicing produces a novel cyclin D1 transcript.

Using Northern blotting and PCR analysis of cDNA derived from a range of cell lines and tissues, alternate splicing of the cyclin D1 gene (CCND1) mRNA has been demonstrated. The variant transcript shows no splicing at the downstream exon 4 boundary, encoding a protein with an altered carboxy-terminal domain. Investigation of mRNA extracted from mononuclear cells, lung tumour and normal tissue suggests that both transcripts are invariably expressed. However, splicing to produce the two forms of mRNA is modulated, in the heterozygote, by a frequent A/G polymorphism located within the splice donor region of exon 4. Preliminary analysis of patients with resectable non-small cell lung cancer suggests that genotype is associated with shortened event free survival and greater risk of local relapse.

Aged↗
PubMed2002

Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays. These NMDA mRNA isoforms are distributed, in vivo, in a region-specific manner in rat brain, such that high levels of exon 21 selection and exon 5 skipping coincide with high NAPOR mRNA expression in the forebrain. Within the forebrain, this spatial correspondence is most striking in the visual cortex. In contrast, low NAPOR expression coincides with the reciprocal pattern of alternative splicing in the hindbrain. Complementary experiments demonstrate a tissue-specific distribution of NAPOR, CUG-BP, and other highly related proteins within the nervous system as assayed by probing forebrain and hindbrain nuclear extracts with monoclonal antibody, mAb 3B1. Thus, NAPOR may be one of a group of closely related proteins involved in splicing regulation within the brain. An intronic RNA element responsible for the silencing of exon 21 splicing is identified by mutational analysis and shown to bind directly to recombinant NAPOR protein, suggesting a model in which exon 21 selection is positively regulated by an antirepression mechanism of action.

Alternative Splicing↗
PubMed1995

The alternatively spliced type II corticotropin-releasing factor receptor, stably expressed in LLCPK-1 cells, is not well coupled to the G protein(s).

Two alternatively spliced corticotropin-releasing factor receptor (CRF-R) cDNAs, type I and type II, were recently isolated from a human cDNA library. The two cDNAs are identical except that the type II cDNA encodes an additional 29 amino acid inserted in the first putative cytoplasmic loop. Since the first cytoplasmic loop is highly conserved in all the members of the hCRF receptor family we have examined whether the presence of the 29 amino acid cassette in CRF-RII influences G protein coupling in LLCPK-1 cells stably expressing the type I and type II hCRF receptors. Whether measured in intact cells or in membrane preparations, LLCPK-1 cells stably expressing CRF-RII have a 4-5 fold lower binding affinity. Maximal CRF-stimulated cAMP accumulation in LLCPK-1 cells stably expressing CRF-RI was 10-15-fold higher than that in LLCPK-1 cells expressing CRF-RII. The EC50 for CRF-stimulated cAMP accumulation in hCRF-RI-expressing cells was in the range of 0.5 +/- 0.2 nM. In contrast, the EC50 for CRF-stimulated cAMP accumulation in hCRF-RII expressing cells was 7.7 +/- 0.2 nM. hCRF increased phosphoinositide turnover in LLCPK-1 cells stably expressing CRF-RI but not in those expressing CRF-RII; this effect required hCRF concentrations of 100 nM and higher. In membrane preparations, GTP-gamma-S inhibited hCRF binding to CRF-RI and shifted the binding Kd from 4.5 nM to 16.7 nM. Conversely, GTP-gamma-S did not influence hCRF binding to CRF-RII in broken cell membranes. Additionally, CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RI was potentiated by GTP, whereas CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RII was insensitive to GTP. These data indicate that CRF-RII is not well coupled to the G protein. Since the only difference between the CRF-RII and CRF-RI is the insert in the first putative cytoplasmic loop, these data indicate that the first cytoplasmic loop plays a crucial role in hCRF receptor coupling to the G protein.

Alternative Splicing↗
PubMed1997

Polymorphisms and intron sequences flanking the alternatively spliced 8-amino-acid exon of gamma2 subunit gene for GABAA receptors.

Gamma-amminobutyric acid (GABA) is a major inhibitory neurotransmitter. Two alternatively spliced forms of the gamma2 subunit of GABAA receptor (gamma2L and gamma2S), which differ by an exon of eight amino acids, show different sensitivities to modulatory effects of ethanol on receptor activities. A 2.7 kb DNA fragment and an 1.7 kb DNA fragment covering respectively the introns upstream and downstream from the 8-amino-acid exon were obtained through PCR-amplification of human genomic DNA using primers derived from cDNA sequences. Total sequencing of these fragments showed a composite 4.2 kb segment containing the 8-amino-acid exon and consensus sequences for RNA splice junctions. Restriction fragment length polymorphisms (RFLP) based on NciI restriction digestion were found among Chinese in Taiwan. This RFLP provides a useful DNA marker for allelic association or linkage analyses of the role of GABAA receptors in predisposition to alcoholism or other neuropsychiatric disorders.

Alternative Splicing↗
PubMed1996

Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms.

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.

3T3 Cells↗