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Results for “Alternative Splicing”

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PubMed1998

Short alternative splice transcripts of the mdm2 oncogene correlate to malignancy in human astrocytic neoplasms.

The mdm2 oncogene encodes a 90-kDa nuclear phosphoprotein that binds and inhibits the function of the p53 tumor suppressor protein. It was recently reported that the expression of alternatively spliced variants of mdm2 correlated with malignancy in ovarian tumors and bladder carcinomas. We analyzed the presence of alternatively spliced mdm2 variants and studied their correlation to p53 status in a total of 66 human astrocytic tumors, including 32 glioblastomas multiforme, 17 anaplastic astrocytomas, 12 astrocytomas, and 5 pilocytic astrocytomas, using a specific nested reverse transcription-PCR technique. The full-length mdm2 transcript was demonstrated in all of the cases. Multiple-sized PCR products were found in 29 cases. Two of 5 pilocytic astrocytomas (40%), none of 12 astrocytomas, and 5 of 17 anaplastic astrocytomas (29%) showed alternative splice variants. In contrast, 22 of 32 glioblastomas (69%) showed the presence of splice variants, demonstrating a significantly higher frequency than in lower-grade astrocytomas (P < 0.0003). A majority of the splice variants were 707 base-type (mdm2-b), which was confirmed by sequence analysis. There was no apparent correlation of the presence of mdm2 splice variants with p53 gene status. These results suggest a new role for mdm2, independent of p53 gene status, as an oncogene in the development of malignant astrocytic tumors.

Alternative Splicing↗

PubMed1994

Targeting of G alpha i2 to the Golgi by alternative spliced carboxyl-terminal region.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) may participate in membrane traffic events. A complementary DNA (cDNA) was isolated from a mouse pituitary cDNA library that corresponded to an alternatively spliced form of the gene encoding the G protein alpha subunit G alpha i2. The cDNA was identical to that encoding G alpha i2 except that the region encoding for the carboxyl-terminal 24 amino acids was replaced by a longer region encoding 35 amino acids that have no sequence similarity with G alpha i2 or other members of the G protein family. This alternative spliced product and the corresponding protein (sGi2) were present in several tissues. Specific antibodies revealed that sGi2 was localized in the Golgi apparatus, suggesting a role in membrane transport. Thus, alternative splicing may generate from a single gene two G protein alpha subunits with differential cellular localization and function.

Alternative Splicing↗

PubMed2006

Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): a possible modulator of nucleotidase activity and purinergic signaling.

Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface, membrane-bound enzyme that hydrolyzes extracellular nucleotides, thereby modulating purinergic signaling. An alternatively spliced variant of NTPDase3 was obtained and analyzed. This alternatively spliced variant, termed "NTPDase3beta", is produced through the use of an alternative terminal exon (exon 11) in place of the terminal exon (exon 12) in the full-length NTPDase3, now termed "NTPDase3alpha". This results in an expressed protein lacking the C-terminal cytoplasmic sequence, the C-terminal transmembrane helix, and apyrase conserved region 5. The cDNA encoding this truncated splice variant was detected in a human lung library by PCR. Like the full-length NTPDase3alpha, the alternatively spliced NTPDase3beta was expressed in COS cells after transfection, but only the full-length NTPDase3alpha is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3beta was co-transfected with full-length NTPDase3alpha, there was a significant reduction in the amount of NTPDase3alpha that was properly processed and trafficked to the plasma membrane as active enzyme, indicating that the truncated form interferes with normal biosynthetic processing of the full-length enzyme. This suggests a role for the NTPDase3beta variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3alpha and NTPDase3beta.

Adenosine Triphosphatases↗

PubMed2000

Alternative splicing of the neurotrophin-3 gene gives rise to different transcripts in a number of human and rat tissues.

The mouse neurotrophin-3 (NT-3) gene has been shown to contain two exons (exon 1A and exon 1B) upstream of the single coding exon (exon 2). These upstream exons are alternatively spliced to the coding exon, generating two different NT-3 transcripts. We investigated whether alternative splicing of two upstream exons also occurs in the human and rat NT-3 gene. It was found that the human and rat NT-3 gene also contains two exons upstream of the main coding exon and that alternative splicing of these upstream exons generates two different NT-3 transcripts : transcript 1A and transcript 1B (TR1B). These two transcripts were widely expressed in several human and rat tissues. Also, a third transcript, transcript 1A1C, derived from splicing of exon 1A to exon 1B before splicing to the coding exon was seen in a small number of rat tissues. Previous quantification of neurotrophin-3 mRNA has not been transcript-specific. Here we describe a transcript-specific semiquantitative RT-PCR method allowing the quantification of TR1B in human tissues. We show that this is the major NT-3 transcript and that expression of this transcript was much higher in the adult when compared with the corresponding fetal tissues.

Adult↗

PubMed1990

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.

Animals↗

PubMed1999

Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development.

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

Alternative Splicing↗

PubMed1995

TGF-beta regulates expression of tenascin alternative-splicing isoforms in fetal rat lung.

Two distinct mRNA-splice isoforms of tenascin (TN) are expressed differentially during rat lung development. The unique temporal expression pattern of two TN isoforms suggests the expression of tenascin is strictly developmentally regulated in rat lung tissue. We investigated molecular mechanisms which modulate alternative-splicing expression of TN in lung development. The effect of transforming growth factor-beta 1 (TGF-beta 1) on regulation of expression of TN isoforms was examined by in vitro lung explant culture. Immunoblotting with anti-TN antibody detected two TN polypeptides in rat lung explant culture, the larger [relative molecular weight (M(r)) 230, TN230] polypeptide and the smaller (M(r) 180, TN180). TGF-beta 1 markedly induced the TN180 isoform and caused only a moderate increase of the TN230 isoform. The effects of TGF-beta 1 were shown to be dose dependent over a physiological range of TGF-beta 1 protein concentration. The induction of TN isoform biosynthesis by TGF-beta 1 was detected 12 h after addition of the growth factor, and the effects endured for up to 48 h at a dose of 5 ng/ml. By reverse transcriptase-polymerase chain reaction through amplification of the entire fibronectin type III (FN-III) splicing domain, two distinct TN isoforms were detected in total RNA isolated from gestational day 21 rat lung explant culture treated with TGF-beta 1 and from postnatal day 8 rat lung. The larger isoform contained five FN-III alternative splicing repeats [1,420 base pairs (bp)], but the shorter splicing isoform lacked four FN-III alternative splicing repeats (340 bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗

PubMed1992

Tissue-specific alternative splicing of the beta-galactoside alpha 2,6-sialyltransferase gene.

The rat alpha 2,6-sialyltransferase gene produces three different sized mRNAs (4.7, 4.3, and 3.6 kilobases (kb)) which exhibit striking tissue-specific expression. Recently, we characterized the cDNA and genomic organization of the 4.3-kb mRNA which is unique to rat liver. In this report cDNAs of the 4.7-kb mRNA found in most tissues and the 3.6-kb mRNA(s) unique to kidney have been cloned and characterized as well as the corresponding genomic sequences which differed from those of the previously characterized 4.3-kb mRNA. The 4.7-kb mRNA was found to be identical to the 4.3-kb mRNA with the exception of two additional exons at the 5'-untranslated end of the transcript. The constitutively expressed 4.7-kb mRNA therefore codes for the same sialyltransferase as the liver-restricted 4.3-kb mRNA. The additional 5'-exons of the 4.7-kb mRNA are located at least 15-40 kb upstream of the promoter responsible for the production of the 4.3-kb liver message. The 3.6-kb mRNA of rat kidney was found to be comprised of three transcripts of similar size. They were only expressed in kidney and were found to be generated from the alpha 2,6-sialyltransferase gene by alternative splicing and alternative promoter utilization. The results reveal the complexity of the alpha 2,6-sialyltransferase gene which produces at least five transcripts via alternative splicing in a tissue-specific fashion.

Amino Acid Sequence↗

PubMed1996

Structure and expression of a plant U1 snRNP 70K gene: alternative splicing of U1 snRNP 70K pre-mRNAs produces two different transcripts.

The product of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K (U1-70K) gene, a U1 snRNP-specific protein, has been implicated in basic as well as alternative splicing of pre-mRNAs in animals. Here, we report the isolation of full-length cDNAs and the corresponding genomic clone encoding a U1-70K protein from a plant system. The Arabidopsis U1-70K protein is encoded by a single gene, which is located on chromosome 3. Several lines of evidence indicate that two distinct transcripts (short and long) are produced from the same gene by alternative splicing of the U1-70K pre-mRNA. The alternative splicing involves inclusion or exclusion of a region (910 bp) that we named "included intron." Two transcripts were clearly detectable in all tissues tested, and the level of the transcripts varied in different organs. The deduced amino acid (427 residues) sequence from the short transcript has strong homology to the animal U1-70K protein and contains an RNA recognition motif, a glycine hinge, and an arginine-rich region characteristic of the animal U1-70K protein. The long transcript has an in-frame translational termination codon within the 910-bp included intron, resulting in a truncated protein containing only 204 amino acids. The protein encoded by the short transcript is recognized by U1 RNP-specific monoclonal antibodies and binds specifically to the Arabidopsis U1 snRNA, whereas the protein from the long transcript does not. In addition, multiple polyadenylation sites were observed in the 3' untranslated region. These results suggest a complex post-transcriptional regulation of Arabidopsis U1-70K gene expression.

Alternative Splicing↗

PubMed1998

Alternatively spliced forms of cyclin D1 modulate entry into the cell cycle in an inverse manner.

Alternative splicing of cyclin D1 gene mRNA has recently been demonstrated. The novel transcript shows no splicing at the downstream exon 4 boundary and encodes a protein with an altered carboxyl-terminal domain that is a cyclin D1 variant; exon 5 is not included in the coding sequence which terminates downstream of exon 4. We here produced cells that exogenously express each form of cyclin D1 and analysed their cell cycle regulation. We found that (1) alternative splicing forms of cyclin D1 modulated entry into the cell cycle in an inverse manner; (2) both splicing forms suppressed cell growth; and (3) cells overexpressing form [a] were inhibited from entry into and completion of the S phase, although form [b]-expressing cells showed no reduction of G1- to S transition. We also found that overexpression of either cyclin D1 form upregulated Rb gene products, suggesting that this upregulation may be one of the causes of growth suppression in cyclin D1 overexpressing cells.

Alternative Splicing↗

PubMed2005

Multiple alternatively spliced transcripts of the receptor tyrosine kinase MuSK are expressed in muscle.

We have cloned and characterized five distinct mouse cDNAs encoding isoforms of MuSK, a receptor tyrosine kinase required for the development of the neuromuscular synapse. Comparison of the cDNA sequences with each other and with the mouse genomic sequence revealed that the cDNAs differ by the presence or absence of three alternatively spliced exons encoding an additional 10, 15 and 8 amino acids respectively. The location and sequences of these exons are conserved in MuSK cDNAs from different species. Examination of mouse genomic sequences revealed that the 15 aa exon sequence is present in a 52 bp exon with a non-canonical 3' splice acceptor site at its 5' end and an internal 3' splice acceptor site consensus 45 bp downstream. Transcripts containing each of the alternatively spliced exons were detected in neonatal and adult mouse muscle, as well as in C2 myotubes. The presence of transcripts encoding MuSK isoforms with distinct extracellular domains in developing mouse muscle suggests that alternative splicing could potentially introduce additional complexity to the activity of MuSK in muscle.

Alternative Splicing↗

PubMed2003

Cloning and initial characterization of an alternatively spliced transcript encoded by the bovine herpes virus 1 latency-related gene.

Bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic sensory neurons of infected cattle. The latency-related (LR) RNA is the only abundantly expressed viral transcript in sensory neurons of latently infected calves. Wild-type expression of LR gene products is required for the latency-reactivation cycle in calves. LR RNA is alternatively spliced in trigeminal ganglia (TG) after infection of calves, suggesting that these alternatively spliced transcripts encode novel factors that regulate specific steps during latency. To begin testing whether these alternatively spliced transcripts have novel functions, the authors cloned a full-length cDNA identified in TG of calves at 7 days post infection (dpi) and compared the functions of this cDNA to the intact LR gene. As a result of splicing, the 7 dpi cDNA contains a novel open reading (ORF) comprised of OFR-2 fused to ORF-1. Overexpression of the 7 dpi cDNA inhibited the BHV-1 immediate-early transcription unit 1 (IEtu1) promoter and the herpes simplex virus type 1 ICP0 promoter. Conversely, the 7 dpi cDNA stimulated the LR promoter in transiently transfected cells. A plasmid containing the LR gene had little effect on IEtu1 or LR promoter activity, indicating that the 7 dpi cDNA has novel functions.

Alternative Splicing↗

PubMed2001

Gene structure and alternative splicing of murine podocalyxin: a member of the CD34 sialomucin family.

Podocalyxin is a sialoglycoprotein of the glomerular podocytes, vascular endothelial cells, platelets, and hemopoietic stem cells. The function of podocalyxin is unknown, but it contains most of the protein bound sialic acid in the glomerulus and is considered vital in the structure and function of the glomerular filtration apparatus. The murine podocalyxin full-length cDNA has been determined and is 5,318 base pairs. The gene localizes to chromosome 6B1 by FISH analysis and contains eight major exons with one additional alternatively spliced exon. The alternatively spliced isoform of podocalyxin codes for a truncated intracellular domain and is expressed in a tissue specific pattern in parallel with full-length podocalyxin. The organization of the gene structure of murine podocalyxin is similar to the murine CD34 gene and suggests a distant evolutionary relationship to CD34.

Alternative Splicing↗

PubMed2002

[A novel alternative splicing isoform of vascular endothelial growth factor gene in human beings].

OBJECTIVE: To investigate a new alternative splicing isoform of vascular endothelial growth factor (VEGF) gene in human bejings. METHODS: The total RNA of the lung tissue of a legally aborted 4-month-old human fetus was isolated and then amplified by RT-PCR. The amplified product was cloned into the pMD18-T plasmid and pcDNA3.1(-). Then sequence analysis was conducted. RESULTS: The electrophoresis of the RT-PCR product showed one short band of VEGF121 comprising of 487 bp and a long band with two fragments: one normal VEGF165 comprising of 619 bp and one fragment comprising of 639 bp which was the same VEGF165 nucleotide sequence with a 20 bp fragment inserted between exon 3 and exon 4. Sequence analysis showed that this 20 bp long nucleotide was inserted from the 3' end of the third intron that contained splicing signal, thus causing shift mutation in reading frame of VEGF gene and early appearance of the stop code UAG in the middle of exon 4. CONCLUSION: A new alternative splicing isoform of VEGF has been identified in the lung tissue of a legally aborted human fetus. Its biological significance remains to be further investigated.

Alternative Splicing↗

PubMed1989

cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

Adenoviruses, Human↗

PubMed2003

Alternative splicing of the muscarinic acetylcholine receptor GAR-3 in Caenorhabditis elegans.

Among the three G-protein-linked acetylcholine (ACh) receptors (GAR-1, -2, and -3) in Caenorhabditis elegans (C. elegans), GAR-3 appears most similar to mammalian muscarinic ACh receptors (mAChRs). The gar-3 gene, unlike mammalian mAChR genes, contains introns within the coding region. In this study, we identified an alternatively spliced isoform of GAR-3 (GAR-3a), which differs only in the third intracellular (i3) loop from the previously described one (GAR-3b). GAR-3a has a 26 amino acid insert in the i3 loop compared with GAR-3b. Reverse transcriptase-polymerase chain reaction (RT-PCR) on stage-specific RNAs indicated that both isoforms are expressed at all developmental stages examined, with gar-3b being more abundantly expressed than gar-3a. When these two GAR-3 isoforms were expressed in Chinese hamster ovary (CHO) cells, they exhibited similar ligand binding characteristics. In response to carbachol treatment, the two isoforms stimulated phosphatidylinositol hydrolysis with similar efficacy. Together with our earlier observations that GAR-1 and GAR-2 undergo alternative splicing, this study shows that alternative splicing plays an important role in promoting molecular diversity of G-protein-linked ACh receptors in C. elegans.

Alternative Splicing↗

PubMed1994

[Presence of alternatively spliced messenger RNA for HLA-A, -B and -C in platelets].

Recently we demonstrated that platelets contain specific messenger RNA (mRNA) encoding for the transmembrane HLA class I molecule (44 kDa). In this study we analyzed lymphocytes as well as platelets for the presence of alternatively spliced mRNA with deleted transmembrane domain resulting in soluble HLA class I antigens (39 kDa). PCR amplification of lymphocyte RNA led to a product specific for alternatively spliced cDNA corresponding to the presence of soluble HLA in plasma, while this product could not be obtained from platelet mRNA. These results suggest that platelets in contrast to lymphocytes are unable to synthesize soluble HLA antigens via alternative splicing.

Alternative Splicing↗

PubMed2006

Bioinformatics of alternative splicing and its regulation.

The sequencing of the human genome and ensuing wave of data generation have brought new light upon the extent and importance of alternative splicing as an RNA regulatory mechanism. Alternative splicing could potentially explain the complexity of protein repertoire during evolution, and defects in the splicing mechanism are responsible for diseases as complex as cancer. Among the challenges that rise in light of these discoveries are cataloguing splice variation in the human and other eukaryotic genomes, and identifying and characterizing the splicing regulatory elements that control their expression. Bioinformatics efforts tackling these two questions are just at the beginning. This article is a survey of these methods.

Alternative Splicing↗