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PubMed2002

Alternative splicing of the rat Ca(v)3.3 T-type calcium channel gene produces variants with distinct functional properties(1).

Molecular diversity in T-type Ca(2+) channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Ca(v)3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Ca(v)3.3 gene produces channels with distinct properties.

Alternative Splicing↗

PubMed1995

A novel alternative splice domain in zebrafish tenascin-C.

We have identified and sequenced clones from a zebrafish library that correspond to tenascin-C (TN-C). The 2036-bp sequence covers the C-terminal segment of the protein. Comparison of this sequence to TN-C from other vertebrates indicates that our sequence starts in FN-III domain 5, continues with one alternative splice domain, then with FN-III domains 6-8 and, finally, the complete fibrinogen-like domain. The alternative splice domain, designated TNfnQ, is not closely identified to any of the domains in other species, and may represent a novel descendent of the alternative splice-domain precursor.

Alternative Splicing↗

PubMed1999

Integrins: alternative splicing as a mechanism to regulate ligand binding and integrin signaling events.

Integrins are a family of transmembrane proteins composed of heterodimers of alpha and beta subunits. With their extracellular domain they bind extracellular matrix proteins or other cell surface molecules, and their cytoplasmic domain binds to cytoskeletal and signaling proteins. Thus, they are in an ideal position to transfer information from the extracellular environment to the interior of the cell and vice versa. For several integrin subunits, alternative splicing of mRNA leads to variations in the sequence of both extracellular and cytoplasmic domains. Many integrin splice variants have specific expression patterns, but for some time, functional differences between these variants were not evident. Recent experiments using transfected cell lines and gene targeting of specific splice variants have contributed significantly to our understanding of the function of these splice variants. The results indicate that alternative splicing is a mechanism to subtly regulate the ligand binding and signaling activity of integrins.

Alternative Splicing↗

PubMed1992

Characterization of the mouse PDGF A-chain gene. Evolutionary conservation of gene structure, nucleotide sequence and alternative splicing.

The mouse platelet-derived growth factor (PDGF) A-chain gene has been structurally characterized and compared with its human counterpart. The organization of the two genes is similar. Both consist of 7 exons spaced by 6 introns of corresponding sizes. As in the human gene, exon 6 encodes a sequence which is alternatively spliced. When present, it codes for an alternative C-terminus of the A-chain. In intron 5, conserved stretches of nucleotides, potentially involved in the regulation of the alternative splicing, are identified. The untranslated sequences show a high degree of nucleotide sequence identity and several conserved consensus binding sites for transcription factors are identified within the 5' untranslated as well as in the 5' flanking region.

Alternative Splicing↗

PubMed2000

Expression analysis and characterization of alternatively spliced transcripts of human IL-7Ralpha chain encoding two truncated receptor proteins in relapsed childhood all.

In the family of cytokines and cytokine receptors, alternative splicing of pre-mRNA is a frequently observed process that generates different protein isoforms from a single genetic locus. The splicing-derived cytokine receptor protein isoforms are mostly soluble receptors or show alterations in their cytoplasmic domain. It is possible that receptor abnormalities or a pathological ratio of different isoforms may contribute to leukaemia by circumventing normal growth factor control or altering the balance of proliferation and differentiation. IL-7 plays a critical role in early stages of both B and T cell maturation. Moreover, it stimulates the expansion of mature T cells including anti-tumour reactive cells as well as a number of T and B cell malignancies underlining its potential importance for deregulated lymphoid proliferation and leukaemogenesis. Here, we present detailed data on the expression of the interleukin 7 receptor alpha chain (IL-7Ralpha) in leukaemic cells from 210 children with acute lymphoblastic leukaemia (ALL) and describe two novel alternatively spliced transcripts of human IL-7Ralpha coding for truncated receptor proteins which are still capable of binding IL-7. IL-7Ralpha mRNA expression was more frequent in more mature pre-B ALL [91% (30/33)] than in common [81% (81/100)] or pro-B ALL [64% (18/28)], or even in T ALL [64% (29/45)]. These results are in concordance with flow cytometric analyses on the proportion of IL-7Ralpha bearing cells among total blast cell population. Our results lead us to assume that splicing derived IL-7Ralpha isoforms play a potential role in modulating IL-7 signal transduction and might be important for the pathogenesis of leukaemia.

Alternative Splicing↗

PubMed1996

An alternative splice variant of human IL-4, IL-4 delta 2, inhibits IL-4-stimulated T cell proliferation.

Alternative splicing of mRNA can generate protein isoforms that are preferentially expressed in different tissues or during different states of cell differentiation or activation. Protein isoforms may have different functions. In this study, we cloned, expressed, and tested functional effects of a naturally occurring splice variant of human IL-4, called IL-4 delta 2. In IL-4 delta 2, the second exon of IL-4 is omitted by alternative splicing, with exons 1, 3, and 4 joined in an open reading frame. We found that IL-4 delta 2 RNA is expressed in the PBMC of all donors tested, usually in lower amounts than IL-4 RNA. In contrast, IL-4 delta 2 RNA is expressed in much higher levels than IL-4 RNA in thymocytes and bronchoalveolar lavage cells, suggesting tissue specificity of expression. IL-4 delta 2 cDNA was expressed in yeast. Recombinant human (rh) IL-4 delta 2 was partially purified and found to be glycosylated, with a protein core of 13 to 15 kDa. Unlike rhIL-4, rhIL-4 delta 2 did not act as a costimulator for T cell proliferation. However, rhIL-4 delta 2 inhibited the ability of rhIL-4 to act as a T cell costimulator. Inhibition was independent of glycosylation and was not mediated by toxicity. Iodinated IL-4 delta 2 was found to bind specifically to human PBMC and tumor lines known to express IL-4 receptors. Excess unlabeled IL-4 inhibited cellular binding of labeled IL-4 delta 2. Thus, rhIL-4 delta 2 is a naturally occurring splice variant of IL-4 that is preferentially expressed in the thymus and airways and inhibits function of complete IL-4. The balance between IL-4 and IL-4 delta 2 may be important in the regulation of IL-4 effects.

Amino Acid Sequence↗

PubMed1995

Alternative splicing of AMPA receptor subunits: regulation in clonal cell lines.

Alternative splicing of AMPA receptors was investigated in rat PC12 and human SH-SY5Y cells. PC12 cells predominantly expressed GluR-B flip mRNA before and after differentiation. In SH-SY5Y cells, each AMPA-receptor subunit showed a distinct splice variant expression profile throughout differentiation. GluR-B mRNA was comparable in expression levels for flip and flop splice variants. In the other AMPA-receptor subunit transcripts the flip form was the more prominent splice variant. After three days post induction a transient elevation of GluR-B and -D flop mRNA expression could be observed.

Alternative Splicing↗

PubMed1994

The PACAP receptor: generation by alternative splicing of functional diversity among G protein-coupled receptors in nerve cells.

Recent molecular characterization of new G protein-coupled receptors (GPCR) draw attention to alternative splicing as a source of structural diversity. After a brief overview of characterized GPCR splice variants, we will describe in more detail the functional properties of the PACAP type I receptor splice variants. Some of these variants are positively coupled to both adenylate cyclase (AC) and phospholipase C (PLC) whereas others do not elicit any stimulation of the PLC or display a qualitatively intermediate phenotype. The PACAP type I receptor is therefore one of the few examples in which alternative splicing is clearly linked to functional diversity.

Alternative Splicing↗

PubMed2006

Hodgkin's lymphoma cells express alternatively spliced forms of HDM2 with multiple effects on cell cycle control.

The HDM2 oncoprotein is a cellular inhibitor of p53 and is frequently deregulated in human cancer. However, the HDM2 gene encodes alternatively spliced variants whose functional significance is poorly understood. We had previously reported the detection of alternative HDM2 forms in Hodgkin's lymphoma (HL)-derived cell lines. Here, we have cloned several of these transcripts, including the previously described HDM2-A, -B and -C (which encode the COOH terminus of HDM2), and two novel variants (HDM2-HL1 and -HL2) containing a complete p53 interaction domain. Real-time PCR assays demonstrated that HDM2-A and -B were selectively expressed by HL cell lines and primary tumors, compared with their non-neoplastic counterparts. In transient transfection experiments, alternatively spliced HDM2 isoforms were partially or totally localized within the cytoplasm. HDM2-HL2 was able to inhibit transactivation of a p53-inducible reporter construct and induced a partial relocalization of p53 to the cytoplasm. Expression of HDM2-A and -B caused the activation of p53/p21 and induced growth arrest in primary cells, but also increased the expression levels of cyclins D1 and E. Other possible genes regulated by HDM2-A and -B were identified using cDNA microarray technology. These results imply that HDM2 isoforms may have multiple effects on cell cycle control, and provide insight into the mechanisms through which these molecules contribute to tumorigenesis.

Alternative Splicing↗

PubMed1998

Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization.

Although ATP-gated ion channel (P2XR) expression is high among anterior pituitary cells, identification of the receptor subtypes and their selective expression within subpopulations of cell types, as well as their physiological role(s), are incompletely characterized. In this study, we focused on the expression and activity of the P2X2R subtype in anterior pituitary cells. Our results indicate that the primary P2X2R gene transcript in pituitary cells undergoes extensive alternative splicing, with generation of six isoforms. Two of these isoforms encode functional channels when expressed in GT1 or HEK293 cells: the wild-type P2X2R and the spliced isoform P2X2-2R, which lacks a stretch of carboxyl-terminal amino acids (Val370-Gln438). Four other clones showed different alterations, including an interfered reading frame starting in the first transmembrane domain and a 27-amino acid deletion in the large extracellular loop. When expressed separately or in combination with wild-type channels, these clones were nonfunctional. In single cell Ca2+ current and cytosolic Ca2+ concentration ([Ca2+)i) measurements, the P2X2R and P2X2-2R had similar EC50 values for ATP and time courses for activation and recovery from desensitization but differed significantly in their desensitization rates. The spliced isoform exhibited rapid and complete desensitization, whereas the wild-type channel desensitized slowly and incompletely. The mRNAs for wild-type and spliced channels were identified in enriched somatotroph, but not gonadotroph or lactotroph fractions. Expression of a functional ATP-gated channel in somatotrophs was confirmed by the ability of ATP to increase the frequency of [Ca2+]i spikes in spontaneously active cells or initiate spiking in quiescent cells. When voltage-gated Ca2+ influx was blocked, ATP increased [Ca2+]i, with a similar profile and EC50 to those observed in GT1 cells heterologously expressing wild-type or spliced P2X2R. The ligand-selectivity profile of native channels was consistent with the presence of P2X2R in somatotrophs. Finally, the desensitization rate of P2X2R in a majority of somatotrophs was comparable to that observed in neurons coexpressing wild-type and spliced channels. These data indicate that alternative splicing of P2X2R and coexpression of P2X2R and P2X2-2R subunits provide effective mechanisms for controlled cationic influx in somatotrophs.

Adenosine Triphosphate↗

PubMed1999

Regulation of fibronectin alternative splicing during peripheral nerve repair.

Wallerian degeneration following peripheral nerve injury is associated with increased production of fibronectin and other extracellular matrix molecules that are thought to enhance repair. We have shown previously that alternative splicing of the mRNA for fibronectin also changes following sciatic nerve lesions so as to reexpress forms of mRNA seen during embryogenesis. In the present study, we have examined the role of the regenerating axons in the regulation of this splicing. We have compared the patterns of fibronectin mRNA splicing seen in sciatic nerve development with that seen in cut nerves (that do not regenerate), crushed nerves (that regenerate successfully), and Schwann cells cultured in forskolin so as to mimic axonal signals. By using a reverse transcriptase polymerase chain reaction assay to examine all three regions of fibronectin mRNA splicing in a quantitative manner, we found that embryonic patterns of fibronectin mRNA splicing appear rapidly following injury and are not then altered by reestablishment of axons in the nerve. In addition, we found that forskolin has no effect on fibronectin mRNA splicing in cultured cells. We conclude that axonal signals do not regulate the pattern of fibronectin alternative splicing in peripheral nerve repair.

Alternative Splicing↗

PubMed2003

Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein.

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.

Alternative Splicing↗

PubMed1993

Neural expression of a novel alternatively spliced and polyadenylated Gs alpha transcript.

We have isolated an alternative transcript of the rat Gs alpha signal transduction protein gene, referred to as Gs alpha N1. Gs alpha N1 was isolated by differential hybridization screening of genes induced upon dexamethasone treatment of the neuronal-like CA77 rat thyroid C-cell line. The 1-kilobase Gs alpha N1 transcript is generated by alternative splicing and polyadenylation of a novel terminal exon. This exon lies 800 base pairs downstream of exon 3 in the Gs alpha gene. Dexamethasone differentially induced Gs alpha N1 severalfold relative to Gs alpha mRNA in the CA77 cells, similar to the bias seen with alternative processing of the calcitonin/calcitonin gene-related peptide transcript. In addition to the differential regulation by dexamethasone, the expression pattern of Gs alpha N1 in rat tissues differed markedly from Gs alpha. Gs alpha N1 mRNA was much more abundant in the brain, with intermediate levels in skeletal muscle and very low levels in other tissues. This was in contrast to the more ubiquitously expressed Gs alpha mRNA. Within the brain, Gs alpha N1 was particularly abundant in discrete regions of the brainstem and hypothalamus that modulate autonomic functions. Examination of rat embryos demonstrated that Gs alpha is expressed in both brain and nonneural tissue at least 1 day before Gs alpha N1 mRNA could be detected in the embryonic brain. Based on the regulated expression of the Gs alpha N1 transcript and previous studies on G alpha proteins, the predicted Gs alpha N1 protein may potentially modulate several heterotrimeric G protein functions in the nervous system.

Alternative Splicing↗

PubMed2003

The birth of an alternatively spliced exon: 3' splice-site selection in Alu exons.

Alu repetitive elements can be inserted into mature messenger RNAs via a splicing-mediated process termed exonization. To understand the molecular basis and the regulation of the process of turning intronic Alus into new exons, we compiled and analyzed a data set of human exonized Alus. We revealed a mechanism that governs 3' splice-site selection in these exons during alternative splicing. On the basis of these findings, we identified mutations that activated the exonization of a silent intronic Alu.

Adenosine Deaminase↗

PubMed1993

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

Amino Acid Sequence↗

PubMed1995

Tissue-specific and developmentally regulated alternative splicing in mouse skeletal muscle ryanodine receptor mRNA.

The ryanodine receptor is a channel for Ca2+ release from intracellular stores. By PCR analysis, we identified two alternatively spliced regions in mRNA of the mouse skeletal muscle ryanodine receptor (sRyR). The splice variants were characterized by the presence or absence of 15 bp (ASI) and 18 bp (ASII) exons. The exclusion of these exons results in the absence of the regions corresponding to Ala3481-Gln3485 and Val3865-Asn3870, respectively, of rabbit sRyR; these amino acid sequences exist in the modulatory region, where sites for phosphorylation and binding of Ca2+, calmodulin and ATP are postulated to be. We also detected sRyR in brain and heart as well as in skeletal muscle, and the splicing patterns were found to be tissue-specific. Only the ASII-lacking isoform was detected in heart, whereas in other tissues the ASII-containing isoform was predominant. The splicing patterns were also found to change during development. In skeletal muscle, the ASI-containing isoform increased gradually from embryo to adult. The ASII-lacking isoform abruptly increased upon birth, but the ASII-containing isoform increased steadily afterwards. In cerebrum, the ratio of the ASII-containing isoform to the ASII-lacking one increased abruptly during embryonic days 14 and 18. These findings suggest that the alternative splicing of ASI and ASII, by affecting the modulatory region, generates functionally different sRyR isoforms in a tissue-specific and developmentally regulated manner.

Alternative Splicing↗

PubMed2001

Alternative splicing of potassium channels: a dynamic switch of cellular excitability.

Alternative splicing of pre-messenger RNA and reversible protein phosphorylation are fundamental mechanisms for regulating protein structure and function. Recent studies of one class of potassium channel (BK(Ca)) reveal dynamic reciprocal interactions between pre-mRNA splicing and protein phosphorylation. Splicing is regulated by phosphorylation, and exon selection determines the sensitivity of the channel protein to regulation by protein phosphorylation. These studies reveal a powerful dynamic molecular switch to determine cellular excitability.

Alternative Splicing↗

PubMed1991

Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.

The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

Amino Acid Sequence↗