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At least 919 records · Page 51Linked to original sources

PubMed1990

Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: alternative coding exons contain inverted repeat sequences.

Polymorphic forms of acetylcholinesterase are tethered extracellularly either as dimers membrane-anchored by a glycophospholipid or as catalytic subunits disulfidelinked to a collagen tail that associates with the basal lamina. Genomic clones of acetylcholinesterase from T. californica revealed that individual enzyme forms are encoded within a single gene that yields multiple mRNAs. Each enzyme form is encoded in three exons: the first two exons, bases -22 to 1502 and 1503 to 1669, encode sequence common to both forms, while alternative third exons encode a hydrophobic C-terminal region, to which a glycophospholipid is added upon processing, and a nonprocessed C-terminus, yielding a catalytic subunit that disulfide-links with a collagen-like structural unit. The 3' untranslated region of each alternative exon contains tandem repeat sequences that are inverted with respect to the other exon. This may either dictate alternative exon usage by formation of cis stem-loops or affect the abundance of translatable mRNA by trans-hybridization between the alternative spliced mRNA species.

Acetylcholinesterase↗

PubMed1995

Evolution of the intron-exon structure of eukaryotic genes.

The origin and evolution of intron-exon structures continue to be controversial topics. Two alternative theories, the 'exon theory of genes' and the 'insertional theory of introns', debate the presence or absence of introns in primordial genes. Both sides of the argument have focused on the positions of introns with respect to protein and gene structures. A new approach has emerged in the study of the evolution of intron-exon structures: a population analysis of genes. One example is the statistical analysis of intron phases--the position of introns within or between codons. This analysis detected a significant signal of exon shuffling in the DNA sequence database containing both ancient and modern exon sequences: intron phase correlations, that is, the association together within genes of introns of the same phase. The results of this analysis suggest that exon shuffling played an important role in the origin of both ancient and modern genes.

Animals↗

PubMed2006

Brain- and heart-specific Patched-1 containing exon 12b is a dominant negative isoform and is expressed in medulloblastomas.

Mutations in the human tumor suppressor gene, Patched-1, are associated with nevoid basal cell carcinoma syndrome characterized by developmental abnormalities and tumorigenesis, such as basal cell carcinoma and medulloblastoma. During the investigation of complex alternative splicing in Patched-1, we identified an alternative exon, exon 12b, located between exon 12 and 13, both in humans and in mice. Since exon 12b has an in-frame stop codon, the mRNA isoform containing this exon (Patched12b) encodes a truncated patched-1 protein. RT-PCR and whole mount in situ hybridization revealed that mouse exon 12b was expressed in the brain and heart, particularly in the cerebellum, in both adults and embryos. We next performed a functional analysis of Patched12b using a GLI-responsive luciferase reporter. Luciferase activity was suppressed when transfected with a plasmid encoding Patched-1, but not with a plasmid for Patched12b. The suppressive activity of Patched-1 was relieved when cotransfected with a plasmid for Patched12b. This implies that the Patched12b protein has a dominant negative effect on Patched-1. Interestingly, Patched12b was found to be expressed in some of the medulloblastoma tissues and cell lines, indicating an important role in the pathogenesis of medulloblastoma as well as brain development.

Alternative Splicing↗

PubMed2005

Intraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle.

Antisense oligodeoxynucleotide against the splicing enhancer sequence (SES) in exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame in the cultured Duchenne muscular dystrophy (DMD) myocytes with the deletion of exon 20. Transfection of the antisense oligodeoxynucleotide, therefore, has been proposed as a promising means for therapeutic modification of dystrophin mRNA of DMD, a fatal disorder caused by defects in the dystrophin gene. A systemic delivery method targeting the large number of diseased muscles remains to be established for clinical application of antisense oligodeoxynucleotide. In this study, we investigated capability of oligodeoxynucleotide transfer into the skeletal muscles of mdx mouse, a mouse model of DMD. Thirty-one mer phosphorothioate oligodeoxynucleotide complementary to the SES of dystrophin exon 19 was intraperitoneally administered to mdx mice without any carrier. Histochemical study disclosed that fluorescence-labeled oligodeoxynucleotide appeared in the nuclei of femoral skeletal muscle cell at the second day after injection of 20 mg/kg BW oligodeoxynucleotide, and still visible at 14th day. Reverse transcription (RT)-PCR analysis of dystrophin transcript in these cells disclosed that a proportion of it showed skipping of exon 19 from second to seventh day after injection. These results showed that the intraperitoneally administered oligodeoxynucleotide could be transfected to nucleus of mdx skeletal muscle without any carrier and was able to induce exon skipping in vivo.

Animals↗

PubMed2005

Deleterious mutations in exon 1 of MECP2 in Rett syndrome.

The MECP2 gene is responsible for 80-85% of typical cases of Rett syndrome with deleterious mutations affecting exons 3 and 4. Recently, an alternate transcript including exon 1 was discovered with a new protein isoform (MeCP2_e1) much more abundant in brain. We screened exon 1 of MECP2 for mutations and for large rearrangements in a panel of 212 typical cases of Rett syndrome and one family case with atypical Rett syndrome. We identified two deleterious mutations (c.48_55dup and c.62+2_62+3del) and four large rearrangements encompassing exon 1 of MECP2. We also identified the c.16_21dup alteration formerly reported as c.3_4insGCCGCC and give additional support to classify this sequence variation as polymorphic. In our large panel of typical Rett, mutations affecting exon 1 of MECP2 represent 1% of the deleterious alleles. This study confirms that mutations in exon 1 of MECP2 are a rare cause of Rett syndrome.

Child, Preschool↗

PubMed2006

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region.

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.

Alternative Splicing↗

PubMed2006

SR protein 9G8 modulates splicing of tau exon 10 via its proximal downstream intron, a clustering region for frontotemporal dementia mutations.

The microtubule-associated protein tau is important to normal neuronal function in the mammalian nervous system. Aggregated tau is the major component of neurofibrillary tangles (NFTs), present in several neurodegenerative diseases, including Alzheimer's and frontotemporal dementia with Parkinsonism (FTDP). Splicing misregulation of adult-specific exon 10 results in expression of abnormal ratios of tau isoforms, leading to FTDP. Positions +3 to +16 of the intron downstream of exon 10 define a clustering region for point mutations that are found in FTDP. The serine/arginine-rich (SR) factor 9G8 strongly inhibits inclusion of tau exon 10. In this study, we established that 9G8 binds directly to this clustering region, requires a wild-type residue at position +14 to inhibit exon inclusion, and RNAi constructs against 9G8 increase exon 10 inclusion. These results indicate that 9G8 plays a key role in regulation of exon 10 splicing and imply a pathogenic role in neurodegenerative diseases.

Alternative Splicing↗

PubMed2006

Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers.

Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between constitutively and alternatively spliced exons. Representatives of these ESRs and two SR protein binding sites were shown, experimentally, to display variable regulatory effects on alternative splicing, depending on their relative locations in the exon. This finding signifies the delicate positional effect of ESRs on alternative splicing regulation.

Alternative Splicing↗

PubMed2003

Arthropod and mollusk defensins--evolution by exon-shuffling.

Arthropod and mollusk defensins are secreted antibacterial proteins that exhibit similarity in sequence, mode of action and structure and are expressed ubiquitously. Comparison of the gene organization of a newly cloned scorpion defensin gene, with that of other arthropods and the mussel, revealed that all exons and introns, aside from the exon encoding the mature protein, differ widely in number, size and sequence. This variability suggests that the exon encoding the mature defensin has undergone exon-shuffling and integrated downstream of unrelated leader sequences during evolution. Unlike other exon-shuffling events, in which modules are added into existing proteins, arthropod and mollusk defensins represent the first instance of exon-shuffling of autonomous modules.

Animals↗

PubMed2005

Influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of SARS-CoV and its implication for subunit vaccine development.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated S protein of the TW1 strain, STR2 (88 kDa), carrying three S fragments (S74-253, S294-739, and S1129-1255) was investigated to study the influences of intron and exon splicing enhancers to improve STR2 protein expression in mammalian cells. Our results showed that STR2 protein expression with the use of an 138 base-pair intron addition increased by 1.9-, 2.5-, and 4.1-fold in Vero E6, QBI-293A cells, and CHO/dhFr- cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. Using the exon splicing enhancers, including a bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), were also found to increase STR2 protein expression in CHO/dhFr- cells by 1.7- and 2.6-fold. Nevertheless, combination of the intron and the exon splicing enhancers resulted in suppressing the intron-enhancing e STR2 protein expression in in CHO/dhFr- cells. Our studies also demonstrated the STR2 protein was mainly as the Endo H-sensitive glycoprotein (115 kDa) expressed in Vero E6, QBI-293A, and CHO/dhFr- cells. However, only a minor form of the Endo H-resistant glycoproteins ( approximately 130 kDa) was detected in CHO/dhFr- cells. Taken together, our results indicated that intron had a better enhancing effect on STR2 protein expression than exon splicing enhancers, and the expression of approximately 130 kDa STR2 glycoprotein was enhanced by the intron addition into the expression vector construct. Results of the present study can provide an optimal strategy to enhance SARS-CoV S protein expression in mammalian cells and may contribute to the development of SARS-CoV subunit vaccine.

Animals↗

PubMed2006

Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2.

Formin family proteins coordinate actin filaments and microtubules. The mechanisms by which formins bind and regulate the actin cytoskeleton have recently been well defined. However, the molecular mechanism by which formins coordinate actin filaments and microtubules remains poorly understood. We demonstrate here that Isoform-Ib of the Formin-1 protein (Fmn1-Ib) binds to microtubules via a protein domain that is physically separated from the known actin-binding domains. When expressed at low levels in NIH3T3 fibroblasts, Fmn1-Ib protein localizes to cytoplasmic filaments that nocodazole disruption confirmed as interphase microtubules. A series of progressive mutants of Fmn1-Ib demonstrated that deletion of exon-2 caused dissociation from microtubules and a stronger association with actin membrane ruffles. The exon-2-encoded peptide binds purified tubulin in vitro and is also sufficient to localize GFP to microtubules. Exon-2 does not contain any known formin homology domains. Deletion of exon 5, 7, 8, the FH1 domain or FH2 domain did not affect microtubule binding. Thus, our results indicate that exon-2 of Fmn1-Ib encodes a novel microtubule-binding peptide. Since formin proteins associate with actin filaments through the FH1 and FH2 domains, binding to interphase microtubules through this exon-2-encoded domain provides a novel mechanism by which Fmn1-Ib could coordinate actin filaments and microtubules.

Actins↗

PubMed2005

Identification and characterization of multiple isoforms of a murine and human tumor suppressor, patched, having distinct first exons.

Mutations in mouse and human patched (PTCH) genes are associated with birth defects and cancer. PTCH, a 12-pass transmembrane protein, is a receptor for Sonic hedgehog (Shh) signaling proteins. Shh proteins activate transcription of target genes, including PTCH, via GLI transcription factors. Here we identified seven and five isoforms of human and mouse PTCH mRNA, respectively, which are generated by the complex alternative use of five exons as the first exon (exons 1a to 1e in the 5'-to-3' order). Although expression profiles of these isoforms were highly variable among human tissues, three of them, PTCHa, PTCHb, and PTCHd, were predominantly expressed in most tissues, PTCHd being most ubiquitous. In contrast, PTCHb was always predominant and reached a maximum at E10.5 during mouse development. These three mRNA isoforms encode three PTCH proteins with distinct N-termini, PTCH(L), PTCH(M), and PTCH(S). The expression of these three isoforms was regulated by GLI transcription factors, and at least two functional GLI-binding sequences were identified, one in exon 1a and the other between exon 1a and exon 1b. PTCH(L) and PTCH(M) were equally active in terms of suppressing GLI-mediated transcription and inducing apoptosis. PTCH(S) protein (encoded by PTCHd), lacking the first transmembrane domain, was more unstable than the other two, resulting in a reduced activity. This study may shed light on the mechanism whereby a single PTCH gene plays a role in both tumor cell growth and embryonic development.

Alternative Splicing↗

PubMed1999

Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia.

In order to explore genomic plasticity at the level of the mini-exon gene-bearing chromosome in natural populations of Leishmania, the molecular karyotype of 84 Leishmania stocks belonging to subgenus Viannia, originating mostly from Peru and Bolivia, and differing according to eco-geographical and clinical parameters, was resolved and hybridised with a mini-exon probe. The results suggest that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245-kb size-difference), and partially involves variation (up to 50%) in copy number of mini-exon genes. There is no significant size-difference between mini-exon-bearing chromosomes of Peruvian and Bolivian populations of cutaneous and mucosal isolates of Leishmania (Viannia) braziliensis, but there is between eco-geographical populations of Leishmania (Viannia) peruviana. Leishmania (V.) peruviana presented a significantly smaller mini-exon-bearing chromosome than the other species of subgenus Viannia. The contrast between the general chromosome size heterogeneity and the homogeneity observed in some Peruvian Andean areas is discussed in terms of selective pressure.

Animals↗

PubMed2001

Sequencing of the coding exons of the LRP1 and LDLR genes on individual DNA samples reveals novel mutations in both genes.

Five coding polymorphisms in de LRP1 gene, i.e. A217V, A775P, D2080N, D2632E and G4379S were discovered by sequencing its 89 exons in three test-groups of 22 healthy individuals, 29 Alzheimer patients and 18 individuals with different clinical and molecularly uncharacterized lipid metabolism problems. No genetic defect was evident in the LRP1 gene of any of the Alzheimer's disease (AD) patients, further excluding LRP1 as a major genetic problem in AD. Lipoprotein receptor related protein (LRP) A217V (exon 6) was clearly present in all groups as a polymorphism, while D2632E was observed only once in a healthy volunteer. On the other hand, LRP1 alleles A775P, D2080N, and G4379 were encountered only in patients with FH or with undefined problems of lipid metabolism. This finding forced one to also analyze the LDL receptor (LDLR) gene, for which a method was devised to sequence the entire region comprising LDLR exons 2-18. The resulting sequence contig of 33567 nucleotides yielded finally an exact physical map that corrects published and listed LDLR gene maps in many positions. In addition, next to known mutations in LDLR that cause FH, four novel LDLR defects were defined, i.e. del e7-10, exon 9 mutation N407T, a 20 bp insertion in exon 4, and a double mutation C292W/K290R in exon 6. No evidence for pathology connected to the LRP1 'mutations' was obtained by subsequent screening for the five LRP1 variants in larger groups of 110 FH patients and 118 patients with molecularly undefined, clinical problems of cholesterol and/or lipid metabolism. In three individuals with a mutant LDLR gene a variant LRP1 allele was also present, but without direct, obvious clinical compound effects, indicating that the variant LRP1 alleles must, for the present, be considered polymorphisms.

Adolescent↗

PubMed2000

Isoform-specific exon skipping in a variant form of ClC-2.

A new form of the widely expressed, volume-regulated chloride channel, ClC-2, has been cloned from a pig ileal cDNA library. This ClC-2 homologue, called ClC-2i, has interesting variability within its cDNA sequence, including the deletion of bases that correspond to positions 1326 through 1401 in rat ClC-2 cDNA sequence. This 75 bp deletion corresponds to the complete loss of exon 13 plus the first four bases of exon 14, and involves an atypical intron-exon splice site. Tissue-specific mRNA expression patterns in the pig show variable degrees of exon 13 skipping in ClC-2i. Exon 13 skipping was also observed in rat ClC-2i, albeit at a higher frequency than in the pig in tissues that were examined. A relatively purine-rich 76 bp insertion in the pig genomic sequence of ClC-2i, close to the 3' end of intron 12, may be responsible for the relatively high frequency of exon 13 skipping during the processing of this mRNA.

Animals↗

PubMed2000

Presence of new alternative exons in human and mouse Fli-1 genes.

The mouse Fli-1 proto-oncogene is activated by proviral integration of four murine leukemia retroviruses and its human counterpart is translocated (11,22) in Ewing tumors. We have identified two alternative exons 1 by RACE analysis from a human neuroectodermal tumor. Exons 1a and 1b are located respectively 1.3 and 2.5 kb upstream from the published exon 1. Translation of these alternative messengers is predicted to generate very similar proteins. The sequence upstream from exon 1b showed functional promoter activity. Exon 1b was not conserved in the mouse but was detected in every analyzed human cell, whereas exon 1a was present only in a subset of them and also in various mouse cell lines. These results suggest that both mouse and human Fli-1 gene expression might be under the control of several independent promoter regions.

Alternative Splicing↗

PubMed1998

A cis-acting regulatory element that affects the alternative splicing of a muscle-specific exon in the mouse NCAM gene.

The pre-mRNA encoding the neural cell adhesion molecule (NCAM) is spliced to generate NCAM isoforms containing the muscle-specific domain (MSD) during myogenesis. Utilizing chimeric NCAM minigenes, we searched for cis-acting elements that contribute to the alternative selection of exon MSDb, one of the four exons encoding MSD, and identified an intronic cis-element located downstream of exon MSDb. The cis-element acted as a negative regulator for the selection of exon MSDb in nonmuscle fibroblasts but not in myoblasts, that are already destined to differentiate into muscle cells. The suppressive effect of this cis-element on the selection of exon MSDb was released in the process of myogenesis. When MyoD was co-expressed with a minigene containing this element in fibroblasts, the suppressive effect of the cis-element was released as the cells underwent differentiation. We propose that this cis-element contributes at least as one of the regulatory elements in the differentiation state-dependent selection of MSD exons in vivo.

Alternative Splicing↗

PubMed1998

Alternative splicing of mouse IL-15 is due to the use of an internal splice site in exon 5.

IL-15 is a pleiotropic cytokine modulating growth and differentiation of several hematopoietic cell types. Recently, we have demonstrated that mouse microglial cells, the brain macrophages, express both IL-15 and IL-15/IL-2 receptors. Based on single-cell RT-PCR data, we describe here an alternatively spliced IL-15 mRNA variant found in a small subpopulation of mouse microglia (5%, 3 out of 60 cells expressing IL-15 transcripts). PCR cycle sequencing of this larger transcript revealed the mouse homologue of the alternatively spliced exon A as it is known from the human IL-15 gene. Analysis of the corresponding mouse IL-15 gene region shows that the larger IL-15 transcript contains an yet unidentified 5' sequence of exon 5 while the shorter transcript uses an internal splice acceptor site. The mouse exon 5A segment has a length of 136 nt (17 nt longer than the human exon A). It contains five in-frame stop codons at its 5' end and a new translation initiation site at its 3' end. This new start site is surrounded by a favourable Kozak consensus sequence suggesting a more efficient translation rate. Further translational control by stem-loop binding factors is inferred by a predicted RNA stem-loop structure around the start site. Insertion of exon 5A would lead to an IL-15 polypeptide with a shortened leader sequence of 26 amino acids, as compared to the 48 amino acid leader sequence encoded by the transcript lacking exon 5A. Thus, the final IL-15 protein of the two splice variants is identical; different leader sequences could, however, lead to differences in the intracellular sorting, processing and/or secretion of IL-15.

Alternative Splicing↗