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Results for “Alternative Splicing”

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PubMed2003

Identification of a novel alternative splicing isoform of human amyloid precursor protein gene, APP639.

Senile plaques, neurofibrillary tangles and amyloid-laden cerebral vessels are characteristic features in the brains of individuals with Alzheimer's disease (AD). The principal component of amyloid in senile plaque and amyloid-laden cerebral vessels is beta-amyloid (Abeta), a peptide proteolytically derived from a large amyloid precursor protein (APP). To date, several alternatively spliced human APP transcripts have been described. Here, we report the identification of a novel alternative splicing isoform of the human APP gene, APP639, which excludes exon 2 as well as exons 7 and 8. RT-PCR and Southern blot analysis show that APP639 mRNA is expressed in many human fetal tissues. In contrast, the APP639 transcript is hardly detected in the aged human cerebral cortex from both pathologically confirmed sporadic AD cases and nondemented controls. However, APP639 mRNA exists in the adult human liver. Western blot analysis shows that the protein product produced from the APP639 cDNA could be recognized by the APP antibody, and it does lack the exon 2 coding region. These results suggest that APP639, a novel alternative splicing isoform of human APP gene, does exist in human tissues in vivo.

Adult↗

PubMed1996

Alternative splicing in COL1A1 mRNA leads to a partial null allele and two In-frame forms with structural defects in non-lethal osteogenesis imperfecta.

We have identified a novel multiexon genomic deletion in one COL1A1 collagen allele that results in three alternative forms of mutant mRNA. This mutation occurs in a 9-year-old girl and her father, both affected with severe type III osteogenesis imperfecta (OI). We previously reported detection of a mismatch in their alpha1(I) amino acids 558-861 region by RNA/RNA hybrid analysis (Grange, D. K., Gottesman, G. S., Lewis, M. B., and Marini, J. C. (1990) Nucleic Acids Res. 18, 4227-4236). Single Strand Conformational Polymorphism further localized the mRNA mutation to the amino acids 579-679 coding region. At the gene level, polymerase chain reaction (PCR) amplification of patient leukocyte DNA from the exon 33-38 region yielded the normal 1004-base pair (bp) fragment and an additional 442-bp fragment. Sequencing of the shorter genomic PCR product confirmed the presence of a 562-bp deletion, extending from the last 3 nucleotides (nt) of exon 34 to 156 nt from the 3'-end of intron 36. The genomic deletion was also detected in the clinically normal grandmother, who was confirmed to be a mosaic carrier. PCR amplification and RNase protection experiments were used to investigate the mRNA structure and occurrence of alternative splicing. One form of the mutant cDNA has a deletion with end points that are identical to the genomic deletion. This results in a combination deletion/insertion, with a deletion of amino acids 603-639 followed by an insertion of 156 nt from the 3'-end of intron 36. In addition, we found two alternatively spliced forms. One form uses a cryptic donor site in exon 34 and the exon 37 acceptor. The second form uses the normal exon 32 splice donor and exon 37 acceptor. Use of the cryptic donor results in a coding sequence that is out-of-frame. Both the retained intron form and the use of the exon 32 donor site result in coding sequences that are in-frame. This is the first report of a collagen defect in OI with alternative splicing generating both in-frame and out-of-frame forms of mRNA. Although the in-frame forms constitute more than 60% of the mRNA from the mutant allele, no mutant protein chain was identified. Collagen produced by cultured OI osteoblasts showed a significant increase in the relative amount of type III collagen but no mutant alpha1(I) chain.

Adult↗

PubMed1996

Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.

We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.

Alternative Splicing↗

PubMed1990

Retroviral expression of alternatively spliced forms of rat fibronectin.

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.

Animals↗

PubMed2000

Alternative splicing in the human interleukin enhancer binding factor 3 (ILF3) gene.

The Interleukin Enhancer Binding Factor 3 (ILF3) gene has been mapped to chromosome 19 in humans and to chromosome 9 in mice. Several reported double-stranded RNA binding proteins including NF90, ILF3, MPP4 and DRBP76 have been suggested to be isoforms of the ILF3 gene but this has not been clearly established. We isolated several ilf3 transcripts from a melanoma cDNA library and two corresponding genomic fragments, and report alternative splicing and polyadenylation site selection in the human ILF3 gene. We show the existence of an alternative splice site responsible for the sequence divergence in the 3' part of the transcripts. Another alternative splicing event at a site between the two double-stranded RNA binding motifs leads to the additional presence in some cases of a four amino acids NVKQ peptide. We also describe the utilization of three distinct polyadenylation signals and the generation of an ilf3 transcript with a long extended 3' UTR. The expression of the different transcripts was evaluated. We used a GenBank sequence for the part of chromosome 19 corresponding to the ILF3 gene to determine the exon-intron organization of the entire gene which spans 38 kb and is divided into 21 exons.

Alternative Splicing↗

PubMed2001

An alternatively spliced HDM2 product increases p53 activity by inhibiting HDM2.

The human counterpart hdm2 of the murine double-minute 2 (mdm2) gene encodes a 90-kD protein (HDM2) that inhibits the function of the p53 tumor suppressor. Hdm2 is amplified in approximately 30% of sarcomas, leading to overproduction of HDM2 and inactivation of p53. Using immunohistochemistry to screen a panel of human tumors for HDM2 overproduction, we detected high levels of HDM2 in the cytoplasm in 25% of lung tumors as opposed to its normal localization in the nucleus. These samples contained full-length hdm2 and several alternate-splice forms of hdm2 mRNA. Sequence analysis revealed deletions in the alternate-splice forms of the p53 binding domain and absence of a nuclear localization signal. In transient transfection assays, one of the alternate-splice forms, HDM2(ALT1), bound and sequestered full-length HDM2 in the cytoplasm. In addition, the binding of HDM2(ALT1) to HDM2 inhibited the interaction of HDM2 with p53, thus enhancing p53 transcriptional activity. These data suggest the existence of another level of regulation of HDM2 which increases the activity of p53.

Alternative Splicing↗

PubMed2002

Structure and evolution of neurexin genes: insight into the mechanism of alternative splicing.

Neurexins are neuron-specific vertebrate proteins with hundreds of differentially spliced isoforms that may function in synapse organization. We now show that Drosophila melanogaster and Caenorhabditis elegans express a single gene encoding only an alpha-neurexin, whereas humans and mice express three genes, each of which encodes alpha- and beta-neurexins transcribed from separate promoters. The neurexin genes are very large (up to 1.62 Mb), with the neurexin-3 gene occupying almost 2% of human chromosome 14. Although invertebrate and vertebrate neurexins exhibit a high degree of evolutionary conservation, only vertebrate neurexins are subject to extensive alternative splicing that uses mechanisms ranging from strings of mini-exons to multiple alternative splice donor and acceptor sites. Consistent with their proposed role in synapse specification, neurexins thus have evolved from relatively simple genes in invertebrates to diversified genes in vertebrates with multiple promoters and extensive alternative splicing.

Alternative Splicing↗

PubMed2002

Cloning and characterization of glucose transporter 11, a novel sugar transporter that is alternatively spliced in various tissues.

We have cloned and characterized a novel glucose transporter (GLUT11) that is alternatively spliced. The GLUT11 gene maps to chromosome 22q11.2 and consists of 13 exons. The long form (GLUT11-L) cDNA uses 13 exons to produce a protein containing 503 amino acids. The short form of GLUT11 (GLUT-11) cDNA is missing exon 2 and produces a protein of 496 amino acids with a 14 amino acid N-terminal difference compared to the long form. GLUT11 has significant similarity to known GLUTs and contains 12 putative membrane-spanning helices along with sugar transporter signature motifs that have previously been shown to be essential for transport activity. The putative glycosylation site of GLUT11 is present in loop 1. Northern blot analysis showed that GLUT11 mRNA is expressed in a number of tissues and most abundantly in the skeletal muscle and heart. RT-PCR assay showed that GLUT11 is alternatively spliced and the two isoforms are distributed differently in various tissues. Immunofluorescence microscopy demonstrated that GLUT11-L resides on the plasma membrane when overexpressed in HEK293T cells. Western blot analysis revealed that GLUT11-L runs as a broad band of approximately 42 kDa that was converted to a 38 kDa polypeptide by PNGase F digestion. Furthermore, a liposome reconstitution functional assay showed that GLUT11-L has glucose transport activity.

Alternative Splicing↗

PubMed1989

Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues.

Alternative splicing of fibronectin pre-mRNA at two distinct regions, termed ED-A and IIICS, was investigated with human adult and fetal tissues by the nuclease S1 protection assay. A clear tissue specificity was observed in the splicing pattern at the ED-A region. More ED-A+ than ED-A- mRNAs were identified in lung, whereas ED-A- mRNAs were predominantly expressed in liver. Endometrium contained nearly equal amounts of ED-A+ and ED-A- mRNAs. The splicing pattern at the ED-A region was also different between adult and fetal liver but not between adult and fetal lung. Tissue type specific splicing was also observed at the IIICS region. Although the mRNA species containing the complete IIICS sequence comprised 40-65% of the total fibronectin mRNAs irrespective of tissue types, expression of the mRNA species lacking a part or all of the IIICS sequence was more pronounced in adult liver than in other tissues including fetal liver. These results strongly suggest that the alternative splicing of fibronectin pre-mRNA in vivo is regulated in a tissue type specific manner at both the ED-A and IIICS regions and that it is developmentally regulated in liver but not in lung. On the basis of these and other observations reported previously, a possibility that a part of the fibronectins synthesized and secreted by hepatocytes is deposited in the tissue matrix is discussed.

Adult↗

PubMed1997

Alternative splicing in the pore-forming region of shaker potassium channels.

We have cloned cDNAs for the shaker potassium channel gene from the spiny lobster Panulirus interruptus. As previously found in Drosophila, there is alternative splicing at the 5' and 3' ends of the coding region. However, in Panulirus shaker, alternative splicing also occurs within the pore-forming region of the protein. Three different splice variants were found within the P region, two of which bestow unique electrophysiological characteristics to channel function. Pore I and pore II variants differ in voltage dependence for activation, kinetics of inactivation, current rectification, and drug resistance. The pore 0 variant lacks a P region exon and does not produce a functional channel. This is the first example of alternative splicing within the pore-forming region of a voltage-dependent ion channel. We used a recently identified potassium channel blocker, kappa-conotoxin PVIIA, to study the physiological role of the two pore forms. The toxin selectively blocked one pore form, whereas the other form, heteromers between the two pore forms, and Panulirus shal were not blocked. When it was tested in the Panulirus stomatogastric ganglion, the toxin produced no effects on transient K+ currents or synaptic transmission between neurons.

Amino Acid Sequence↗

PubMed2001

Alternative splicing and sequence diversity of transcripts from the oncosphere stage of Taenia solium with homology to the 45W antigen of Taenia ovis.

Genes and transcripts which show homology to the host-protective 45W antigen of Taenia ovis have been cloned from the human parasite Taenia solium. The T. solium genes cloned in this study (TSO45) show conserved genomic structural features which are also features of the T. ovis 45W gene family. The TSO45 genes consist of a four exon and three intron structure. Eight TSO45 transcripts, encoded by at least five genes, were cloned from T. solium oncospheres and comparison of their DNA sequence indicates that some transcripts have arisen by alternative splicing, the first demonstration of exon inclusion/exon skipping in cestodes. Alternative splicing occurred with respect to both exons II and III with three splice variants identified from the TSO45-1 gene and two splice variants from TSO45-5. The proteins encoded by this family of genes contain putative N-linked glycosylation sites, an amino terminal secretory signal, a hydrophobic carboxy terminal sequence characteristic of GPI-anchored proteins and fibronectin type III motifs. These features are common to their T. ovis and Taenia saginata homologues. The similarities of the TSO45 genes cloned in this study with genes encoding host-protective antigens of T. ovis and T. saginata indicates that the encoded T. solium proteins are quite possibly antigenic and have potential use as a vaccine to prevent T. solium infection in the parasite's intermediate host. In this respect, the generation of sequence diversity and hence potential antigenic diversity through alternative splicing of TSO45 genes may have implications for the use of these proteins in vaccines against T. solium cysticercosis.

Alternative Splicing↗

PubMed1990

Alternative splicing of endothelial cell fibronectin mRNA in the IIICS region. Functional significance.

Transforming growth factor-beta 1 (TGF-beta 1) is thought to play a role in modulating vascular cell function in vivo. In vitro, it decreases endothelial cell proliferation and migration. We postulated that these biologic activities could be mediated through TGF-beta 1 modulation of specific gene expression. Therefore we differentially screened a human umbilical vein endothelial cell cDNA library with cDNAs prepared from both untreated and TGF-beta 1-treated bovine aortic endothelial cells. Using this technique, we isolated many TGF-beta 1-induced cDNA clones. Sequence analysis of these cDNAs showed that many of them corresponded to alternatively spliced fibronectin mRNAs. These fibronectin clones all contained the extradomain I (ED I) but three different forms of the type III connecting segment (IIICS). These different fibronectin cDNAs were expressed in bacteria and the recombinant proteins used to study the effects of IIICS alternative splicing on cell attachment, spreading, and migration in bovine aortic endothelial and smooth muscle cells and B16F10 melanoma cells. The results of these experiments show that attachment and spreading of bovine aortic endothelial and smooth muscle cells depend primarily on the presence of the Arg-Gly-Asp-Ser (RGDS) sequence in the recombinant fibronectin proteins. However attachment and spreading of bovine aortic endothelial cells are modulated by alternative splicing in the IIICS region. Specifically splicing of the IIICS region decreases spreading and increases migration rates of the endothelial cells. On the contrary, using a cell line (B16F10 melanoma cells) that is known not to require the RGDS sequence for adhesion confirmed previous findings that B16F10 melanoma cells do not require the presence of the RGDS sequence for attachment and spreading. Indeed B16F10 cells were able to attach and spread on two recombinant proteins that did not contain the RGDS sequence. However attachment and spreading of B16F10 were dramatically inhibited when a 75-base pair DNA fragment was removed from the 5' end of the IIICS region. These results suggest that various regions of the fibronectin molecule may be able to interact with different cell populations to promote cell attachment and spreading, and that alternative splicing may modulate this process.

Animals↗

PubMed2001

Caspase-2 pre-mRNA alternative splicing: Identification of an intronic element containing a decoy 3' acceptor site.

We have established a model system using the caspase-2 pre-mRNA and initiated a study on the role of alternative splicing in regulation of programmed cell death. A caspase-2 minigene construct has been made that can be alternatively spliced in transfected cells and in nuclear extracts. Using this system, we have identified a 100-nt region in downstream intron 9 that inhibits the inclusion of the 61-bp alternative exon. This element (In100) can facilitate exon skipping in the context of competing 3' or 5' splice sites, but not in single-intron splicing units. The In100 element is also active in certain heterologous pre-mRNAs, although in a highly context-dependent manner. Interestingly, we found that In100 contains a sequence that highly resembles a bona fide 3' splice site. We provide evidence that this sequence acts as a "decoy" acceptor site that engages in U2 snRNP-dependent but nonproductive splicing complexes with the 5' splice site of exon 9, hence conferring competitive advantage to the exon-skipping splicing event (E8-E10). These results reveal a mechanism of action for a negative intronic regulatory element and uncover a role for U2 snRNP in the regulation of alternative splicing.

3' Untranslated Regions↗

PubMed2002

Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein.

Nedd-4-like proteins are E3 ubiquitin-ligase molecules which regulate key trafficking decisions, including targeting of proteins to proteosomes or lysosomes. Here we show that a human Nedd4 family gene, WWP1, is localized on 8q21 and generates at least six isoforms through alternative splicing. We show that alternative splicing affects the domain structure of WWP1, with forms that contain or lack an N-terminal C2 domain. Interestingly, the relative ratio of these forms varies in a tissue-specific manner. Other splice forms were also identified which may disrupt the structure of the C2 domain by removing its predicted C-terminal beta-strands. One splice form generates, through the introduction of a reading frame shift, a C2 domain-only form of WWP1. We discuss the hypothesis that regulation of splice site usage may modulate the activity of WWP1 and possibly other Nedd4 family proteins.

Alternative Splicing↗

PubMed2006

SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness.

Two splice variants are derived from the caspase-9 gene, proapoptotic caspase-9a and antiapoptotic caspase-9b, by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. Previous studies from our laboratory have shown that the alternative splicing of caspase-9 and the phosphorylation status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and ceramide via the action of protein phosphatase-1. In this study, a link between ceramide, SR proteins, and the alternative splicing of caspase-9 was established. The downregulation of SRp30a in A549 cells by RNA interference technology resulted in an increase in the caspase-9b splice variant, with a concomitant decrease in the caspase-9a splice variant, thereby significantly decreasing the caspase-9a/9b ratio from 1.67 +/- 0.11 to 0.56 +/- 0.08 (P < 0.005). The specific downregulation of SRp30a also inhibited the ability of exogenous ceramide treatment to induce the inclusion of the exon 3, 4, 5, and 6 cassette. Therefore, we have identified SRp30a as an RNA trans-acting factor that functions as a major regulator of caspase-9 pre-mRNA processing and is required for ceramide to mediate the alternative splicing of caspase-9.

Alternative Splicing↗

PubMed2004

Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene.

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C beta (PKCbeta) isozymes and preferentially switches the expression to PKCbetaII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCbetaII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCbetaII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCbetaII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCbetaII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.

Adipocytes↗

PubMed2006

Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. RESULTS: By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. CONCLUSION: The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

Alternative Splicing↗

PubMed2005

Alternative splicing and promoter use in the human GABRA2 gene.

GABA(A) receptors mediate the majority of the fast synaptic inhibition in the mammalian brain. They are the targets of several important drugs, including benzodiazepines, which are used as anxiolytics, sedatives, anti-convulsants, and in the treatment of alcohol withdrawal symptoms. Non-coding variations in GABRA2, the gene encoding the alpha2 subunit, are associated with the risk for alcoholism, suggesting that regulatory differences are important. GABRA2 mRNAs from whole human brain and from three brain regions were examined for evidence of alternative splicing using reverse transcription-PCR and DNA sequencing. A complex pattern of alternative splicing and alternative promoter use of the human GABRA2 mRNA was demonstrated. There are four major isoforms consisting of combinations of two alternative 5' and 3' exons, as well as minor isoforms lacking exon 4 or exon 8. The alternative 5' exons each lie downstream of a functional promoter sequence, as shown by transient transfection assays. The promoter activities of naturally occurring haplotypes differed, indicating genetic differences in gene expression.

Alcoholism↗