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PubMed2005

A single glutamine synthetase gene produces tissue-specific subcellular localization by alternative splicing.

Glutamine synthetase (GS) plays a key role in two major biochemical pathways: In liver GS catalyzes ammonia detoxification, whereas in neural tissues it also functions in recycling of the neurotransmitter glutamate. In most species the GS gene gives rise to a cytoplasmic protein in both liver and neural tissues. However, in species that utilize the ureosmotic or uricotelic system for ammonia detoxification, the enzyme is cytoplasmic in neural tissues, but mitochondrial in liver cells. Since most vertebrates have a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in the ureosmotic elasmobranch, Squalus acanthias (spiny dogfish), two different GS transcripts are generated by tissue-specific alternative splicing. The liver transcript contains an alternative exon that is not present in the neural one. This exon leads to acquisition of an upstream in-frame start codon and formation of a mitochondrial targeting signal (MTS). Therefore, the liver product is targeted to the mitochondria while the neural one is retained in the cytoplasm. These findings present a mechanism in which alternative splicing of an MTS-encoding exon is used to generate tissue-specific subcellular localization.

Alternative Splicing↗

PubMed1999

Identification of three alternatively spliced variants of human CD28 mRNA.

CD28, expressed by T cells, plays a central role in providing costimulatory signals to T cells. The cd28 gene is organized into 4 exons. An alternatively spliced CD28 mRNA lacking most of the exon 2 has been previously evidenced. We report here that non stimulated human T cells express three additional alternatively spliced variants of CD28 mRNA (CD28a-c) in. The CD28a variant, expressed at similar levels to that of the full length CD28 mRNA encoding for the membrane form, lacks exon 3. This deletion introduces (i) a frame shift resulting in the addition of two extra amino acids and a premature stop codon and, (ii) induces the loss of the transmembrane region, suggesting that it could encodes for a soluble monomeric molecule which conserves the binding sites of CD28. The CD28b and CD28c variants, expressed at a low level compared with CD28a, are generated by deletion of most of the 3' end of exon 2 plus exon 3 and exon 2 plus exon 3, respectively. Activated T cells express only the membrane CD28 mRNA. These results suggest that resting human T cells may constitutively express both membrane and soluble CD28 which can differentially regulate the outcome of the T cell response.

Alternative Splicing↗

PubMed2000

Alternative splicing forms of the human CD1D gene in mononuclear cells.

CD1d is a critical molecule for the presentation of lipid antigens to natural killer (NK) T cells. To investigate the molecular complexity of CD1d, alternatively spliced transcripts in peripheral blood mononuclear cells from three healthy subjects were analyzed by PCR and sequencing methods. We found eight alternatively spliced variants of the CD1D gene (V1-V8), seven of which are newly established variants (V2-V8). V1 and V4 are in-frame; however, the other six variants (V2, V3, V5-V8) are out-of-frame. V1, V2, V4, and V5 lack a beta(2)-microglobulin binding site (alpha3 domain), indicating the unstable presentation of the CD1d molecule on the surface. In V2 and V5, the transmembrane region is absent, supporting a soluble CD1d. In the V3-V8 variants, the antigen binding region (alpha1 and alpha2 domains) is partially defective, suggesting incomplete functional products. In contrast, the V1 and V2 transcripts bear the complete antigen binding site, resulting in functional proteins. Especially, the V2 splicing variant might function as an inhibitory soluble CD1d molecule and regulate the presentation of antigens on APC to NKT cells.

Alternative Splicing↗

PubMed2005

Alternative splice variants of the human PD-1 gene.

PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Deltaex2, PD-1Deltaex3, PD-1Deltaex2,3, and PD-1Deltaex2,3,4) in addition to the full length isoform. PD-1Deltaex2 and PD-1Deltaex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out. PD-1Deltaex3 is therefore likely to encode a soluble form of PD-1. PD-1Deltaex2,3 lacks exon 2 and 3. These three variants have unaffected open reading frames. PD-1Deltaex2,3,4 lacks exon 2, 3, and 4 (intracellular domain) and contains a premature stop codon in exon 5. Activation of human PBMCs with anti-CD3+anti-CD28 monoclonal antibodies induces an increased level of each PD-1 transcript. A parallel increase in the expression of PD-1Deltaex3 and flPD-1 upon activation suggests an important interplay between the putative soluble PD-1 and flPD-1 possibly involved in maintenance of peripheral self-tolerance and prevention of autoimmunity.

Alternative Splicing↗

PubMed1997

Increased proportion of exon 9 alternatively spliced CFTR transcripts in vas deferens compared with nasal epithelial cells.

CFTR transcripts have been qualitatively and quantitatively analysed in nasal epithelial and vas deferens cells by means of reverse transcription PCR. Alternative splicing of exon 9, which is known to occur in nasal epithelial cells, also occurred in vas deferens cells. The extent of this alternative splicing was determined by the allele present at the Tn locus at the end of intron 8 of the CFTR gene. However, the proportion of transcripts lacking exon 9 sequences was increased in vas deferens cells compared with nasal epithelial cells, independent of the Tn genotype. We postulate that this tissue specific difference in the proportion of CFTR transcripts lacking exon 9 sequences could contribute to the tissue specific disease phenotype observed in individuals with congenital bilateral absence of the vas deferens.

Alternative Splicing↗

PubMed1991

Alternative splicing is an efficient mechanism for the generation of protein diversity: contractile protein genes as a model system.

Alternative splicing has emerged in recent years as a widespread device for regulating gene expression and generating protein diversity. Its analysis has provided some mechanistic understanding of this form of gene regulation and, in addition, has provided new insights into some fundamental aspects of splicing. This mode of regulation is particularly prevalent in muscle cells, where genes such as troponin T are able to generate up to 64 different isoforms from a single transcriptional unit. Alternative splicing has the potential to raise the coding capacity of the small multigene families that code for the contractile proteins so that several million structurally different sarcomeres can be generated. The mammalian alpha-tropomyosin gene has proved particularly useful for the analysis of the mechanisms involved in this type of regulation. In particular, the mutually exclusive splicing of exons 2 and 3 has provided answers about the processes involved in the three main regulatory steps: (a) establishment of mutually exclusive behavior; (b) the elements involved in setting up the default pattern of splicing, and (c) the switch from the default to the regulated splicing pattern in some cell types.

Animals↗

PubMed1990

Molecular diversity of L-type calcium channels. Evidence for alternative splicing of the transcripts of three non-allelic genes.

The diversity of L-type calcium channels was probed using the polymerase chain reaction and primers based on regions conserved in the L-type skeletal muscle (CaCh 1) and cardiac calcium channels (CaCh 2). Related sequences were amplified from human heart, hamster heart, rabbit heart, mouse ovary, mouse BC3H1 cells, and hamster insulin-secreting (HIT) cells. Sequencing of various clones revealed the presence of alternate splicing in gene products coding for CaCh 1, CaCh 2, and a related calcium channel. This related gene product, which we refer to as neuroendocrine or CaCh 3, is expressed in brain and endocrine cells. The diverse products can be explained by the use of alternate exons of equal size, which account for changes in amino acid composition, in combination with an alternate splice acceptor site or an exon skipping event, which produces channels of variable length. Four variants were defined for the gene 3 product, subtypes 3a, 3b, 3c, and 3d that differed in both the sequence of the third membrane spanning segment of the fourth repeat unit (IVS3) and in the size of the linker between this and the fourth membrane spanning segment (IVS4). Three CaCh 2 variants were cloned, subtypes 2a, 2c, and 2d, that are homologous to the a, c, and d variants of CaCh 3. For the skeletal muscle calcium channel only two variants were isolated. They are homologous to those of the a and c subtypes of CaCh 2 or 3, in that they differ only in the size of the IVS3 to IVS4 linker. These results demonstrate that calcium channel diversity is created by both the expression of distinct genes and the alternate splicing of these genes.

Alleles↗

PubMed1997

Analysis of the 5' sequence, genomic structure, and alternative splicing of the presenilin-1 gene (PSEN1) associated with early onset Alzheimer disease.

Mutations in the human presenilin genes (PSEN1 and PSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5' upstream sequence and the structure of the PSEN1 gene reveals that the 5' sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5'UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem-loop structure with stabilities greater than -16 kcal/mol.

Alternative Splicing↗

PubMed1988

Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo.

A single rat gene encodes both fibroblast TM-1 and skeletal muscle beta-tropomyosin by an alternative RNA-processing mechanism. The gene contains 11 exons: Exons 1-5 and exons 8 and 9 are constitutive exons common to all mRNAs expressed from this gene; exons 6 and 11 are used in fibroblasts as well as smooth muscle; exons 7 and 10 are used exclusively in skeletal muscle. We have studied the internal alternative RNA splice choice (exons 6 and 7) of the rat tropomyosin 1 gene in vitro, using nuclear extracts obtained from HeLa cells. Use of alternative splice sites in vitro is dependent on the ionic conditions of the assay, and correct splicing occurs only under well-defined salt conditions. Splicing of exon 5 to exon 6 (fibroblast-type splice) and exon 5 to exon 7 (skeletal muscle-type splice) was dependent on precursors in which exon 6 or 7 was first joined to exon 8. The same patterns of alternatively spliced RNAs were formed when similar templates were introduced in HeLa cells by transfection. Thus, there appears to be an ordered pathway of splicing in which the internal alternatively spliced exons must first be joined to the downstream constitutive exon before they can be spliced to the upstream constitutive exon. The data are consistent with a model in which the critical event in alternative splicing occurs during the joining of exon 6 to exon 8 (fibroblast-type splice) or exon 7 to exon 8 (skeletal muscle-type splice).

Animals↗

PubMed1996

Extracellular pH controls pre-mRNA alternative splicing of tenascin-C in normal, but not in malignantly transformed, cells.

In cultured normal human fibroblasts, 2 main tenascin-C (TN-C) isoforms are generated by alternative splicing of the single TN-C primary transcript, 8 type III repeats being included or omitted in the mRNA. In these cultured cells, small pH variations of the culture medium (from 7.2 to 6.8) strikingly modify the alternative splicing pattern of the TN-C primary transcript. We report that malignantly transformed cells do not respond to extracellular pH variations as normal cells do. Indeed, malignantly transformed cells kept in culture media at pH values from 6.6 to 7.6 show no variations in the splicing pattern of the TN-C primary transcript and accumulate almost exclusively the large TN-C mRNA. These observations may explain the preferential accumulation in vivo of the large TN-C isoform in the extracellular matrix of different types of neoplasia.

Alternative Splicing↗

PubMed1992

Developmental control and alternative splicing of the placentally expressed transcripts from the human growth hormone gene cluster.

Four of the five genes in the human growth hormone gene cluster are expressed in the villous layer of the placenta. We report that the expression of these genes, hCS-A, hCS-B, hCS-L, and hGH-V, are coordinately induced during fetal development, increasing between 12 and 20 weeks of gestation and then plateauing through term. Within the context of this coordinate activation, these genes are expressed at widely different levels and are alternatively spliced in different patterns. There is a developmentally regulated switch in the relative expression of the two chorionic somatomammotropin genes, hCS-A and hCS-B. Starting from approximately equal levels at 8 weeks of gestation, hCS-A is expressed 5-fold more abundantly than hCS-B by term. The proportion of alternatively spliced hGH-V transcripts that retain intron 4 is also developmentally regulated, increasing 3-fold during gestation to 15% at term. A small percentage of hCS transcripts stably retain intron 4 through gestation, the majority derived from the hCS-A gene. hCS-L transcripts undergo two distinct, developmentally stable, splicing pathways between exons 2 and 3. These result from the absence of the normal splice-donor site in intron 2 and the activation of two cryptic splice-acceptor sites. Despite high levels of sequence identity, the four placentally expressed genes in the growth hormone cluster generate a complex set of mRNAs based on alternative splicing and developmental regulation during gestation.

Amino Acid Sequence↗

PubMed2005

The switch in alternative splicing of cyclic AMP-response element modulator protein CREM{tau}2{alpha} (activator) to CREM{alpha} (repressor) in human myometrial cells is mediated by SRp40.

The transcription factor cAMP-response element modulator (CREM) protein, plays a major role in cAMP-responsive gene regulation. Biological consequences resulting from the transcriptional stimuli of CREM are dictated by the expression of multiple protein isoforms generated by extensive alternative splicing of its precursor mRNA. We have previously shown that alternative splicing enables the expression of the CREM gene to be "switched" within the human myometrium during pregnancy from the production of CREMtau(2alpha), a potent transcriptional activator to the synthesis of CREMalpha, a transcriptional repressor. Furthermore we have recently reported that this change in the expression of CREM spliced variants is likely to have important ramifications on the regulation of downstream cAMP-response element-responsive target genes involved in uterine activity during gestation. We have investigated the splicing factors involved in controlling the expression of myometrial CREM splice variants. Data presented here from transient transfections indicate that the switch in the synthesis of CREMtau(2)alpha to CREMalpha that occurs during pregnancy is regulated primarily by an SR protein family member, SRp40. We also show that expression of this splicing factor is tightly regulated in the myometrium during pregnancy. SRp40 regulates the splicing of CREM via its interactions with multiple ESE motifs present in the alternatively exons of CREM. In vitro splicing and electrophoretic mobility shift assays were employed to confirm the functionality of the SRp40-binding ESEs, thus providing a mechanistic explanation of how SRp40 regulates the switch in splicing from production of CREMtau(2)alpha to CREMalpha.

Alternative Splicing↗

PubMed1988

Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene.

Ca2+-ATPase molecules present in the microsomal fraction from non-muscle cells were examined immunologically. Rabbit whole brain, cerebellum, liver, kidney, and COS-1 cell microsomes all displayed a polypeptide of about 110 kDa which was immunoreactive with a polyclonal antiserum against the cardiac muscle sarcoplasmic reticulum Ca2+-ATPase molecule, but was not immunoreactive with a monoclonal antibody specific for the fast-twitch muscle Ca2+-ATPase. cDNAs encoding the full length of two Ca2+-ATPase molecules were isolated from a human kidney library using a mixture of nucleotide probes derived from both rabbit fast-twitch and cardiac muscle Ca2+-ATPase cDNAs. The human kidney cDNAs, HK1 and HK2, are the products of alternative splicing. HK2 codes for a protein identical to rabbit cardiac muscle Ca2+-ATPase, with the exception of 6 scattered amino acid replacements, whereas HK1 codes for a protein identical to that encoded by HK2, but with the carboxyl-terminal 4 amino acids replaced by an extended sequence of 49 amino acids. cDNAs of the HK1 type are by far the most abundant in the library. The partial structure of a 40-kilobase genomic DNA encoding all but the 5' end of the human cardiac Ca2+-ATPase is described. The exons which give rise to the alternatively spliced products were located by Southern blotting and sequencing, and the alternative splicing patterns were determined.

Amino Acid Sequence↗

PubMed1995

Alternative splicing of the tuberous sclerosis 2 (TSC2) gene in human and mouse tissues.

The recently isolated gene for tuberous sclerosis 2 (TSC2) encodes a 5.5-kb transcript that is widely expressed. The TSC2 gene product, named tuberin, is a 1784-amino-acid protein that shows a small stretch of homology to the GTPase activating protein rap1GAP. We have detected a novel variant of the TSC2 mRNA lacking 129 nucleotides, predicting an in-frame deletion of 43 amino acids spanning codons 946-988 of tuberin. This 129-bp deletion precisely corresponds to exon 25 of the TSC2 gene suggesting that alternative splicing leads to production of two forms of transcripts designated isoforms 1 and 2. Further molecular analysis revealed a third isoform exhibiting a deletion of 44 amino acids spanning codons 946-989 of tuberin. Amino acid 989 is a Ser residue encoded by the first codon of exon 26. The two isoforms also exist in newborn and adult mouse tissues, reinforcing the potential functional importance of these alternatively spliced products. These alternative isoforms should have implications for efforts aimed at identifying mutations in TSC patients. The distinct polypeptides encoded by the TSC2 gene may have different targets as well as functions involved in the regulation of cell growth.

Alternative Splicing↗

PubMed2004

Accumulation of spermidine/spermine N1-acetyltransferase and alternatively spliced mRNAs as a delayed response of HeLa S3 cells following X-ray irradiation.

PURPOSE: A key enzyme of polyamine catabolism, spermidine/spermine N(1)-acetyltransferase (SSAT), is responsive to antiproliferative agents. The role of SSAT in cellular responses to X-ray irradiation was examined. MATERIALS AND METHODS: Exponentially growing HeLa S3 cells were irradiated by X-rays, and mRNA levels for SSAT were measured as a function of post-irradiation time through Northern hybridization. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect alternatively spliced SSAT mRNAs. The intracellular polyamine content was measured by the o-phthalaldehyde method and the enzymatic activity of SSAT by the increased amount of acetylated spermidine after incubation. RESULTS: Not only SSAT mRNA, but also an alternatively spliced mRNA accumulated at the initial stage of growth inhibition after the first or second replication of irradiated cells. The maximum fold increase relative to the level of non-irradiated cells was 3.0-3.5 for both transcripts after 5-Gy irradiation. On the other hand, the mRNA of ornithine decarboxylase, a key enzyme of polyamine synthesis, was little influenced by X-ray treatment. Enzymatic activity of SSAT and the acetylspermidine level were elevated after X-ray irradiation. CONCLUSIONS: Activation of SSAT and the induction of alternatively spliced mRNA of the SSAT gene play an important role in regulating growth inhibition and cell death after X-ray irradiation.

Acetyltransferases↗

PubMed1992

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.

Amyloid beta-Protein Precursor↗

PubMed1994

Alternative splicing of the mouse amelogenin primary RNA transcript.

A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5' ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5' untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.

Alternative Splicing↗

PubMed1995

Detection and analysis of an alternatively spliced isoform of interleukin-6 mRNA in peripheral blood mononuclear cells.

Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transciptase-polymerase chain reaction (RT-PCR) studies to examine lL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PCR product, a second 0.45-kb product was observed. Cloning and dideoxy sequence analysis of this product revealed evidence for an alternatively spliced lL-6 transcript lacking exon II. Further RT-PCR analysis using forward primers ending at or one base before the exon I donor splice site again yielded both products. Additional primers were designed and successfully used to selectively distinguish the two forms of IL-6 transcript. Both transcripts were prominent in peripheral blood monocytes and lymphocytes, whereas only the 0.64-kb, full-length transcript was prominent in the lL-6-producing 5637 (human bladder carcinoma) cell line. Northern analysis revealed, in addition to the predominant 1.3-kb transcript, several minor transcripts at 1.9 to 4.8 kb that hybridized with the alternatively spliced cDNA probe but not with an exon II probe. Western analysis revealed lL-6 polypeptides of predicted size (26 to 29 kD) in culture medium from PBMC, while showing an immunoreactive band at 17 kD in cell lysates. These findings suggest the existence of an alternatively spliced form of lL-6 mRNA, which would encode for a polypeptide missing the gp130 interactive (signal-transducing) domain contained in exon II while retaining the lL-6 receptor (p80) domain. Such a molecule could in theory function as a natural antagonist of lL-6, as it would be expected to bind to the IL-6 receptor but not lead to signal transduction.

Amino Acid Sequence↗