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PubMed2006

Splicing analysis disclosed a determinant single nucleotide for exon skipping caused by a novel intraexonic four-nucleotide deletion in the dystrophin gene.

BACKGROUND: Mutations in exonic splicing enhancer sequences are known to cause splicing errors. Although exonic splicing enhancers have been identified as a stretch of purine-rich sequences, it has been difficult to precisely pinpoint the determinant nucleotides in these sequences. This article reports that a 4-bp deletion in exon 38 of the dystrophin gene induced complete exon 38 skipping in vivo. Moreover, the third nucleotide of the deletion was shown to be determinant for the exonic splicing enhancer activity in in vivo splicing analysis of hybrid minigenes encoding mutant exons. METHOD: Genomic DNA analysis of a 2-year-old boy with a raised level of serum creatine kinase yielded a 4-bp deletion 11 bp upstream of the 3' end of exon 38 of the dystrophin gene (c. 5434-5437del TTCA), disrupting a predicted SC35-binding site. RESULT: Interestingly, his dystrophin mRNA was shown to completely lack exon 38 (exon 38- transcript). As the exon 38- transcript coded for a truncated dystrophin protein, this exon skipping was determined to be a modifying factor of his phenotype. In an in vivo splicing assay, a hybrid minigene encoding exon 38 with the 4-bp deletion was shown to induce complete exon 38 skipping, confirming the deleted region as a splicing enhancer sequence. Site-directed mutagenesis of the deleted sequence showed that the complete exon 38 skipping was caused by mutation of the third nucleotide position of the deletion (C5436), whereas mutations at the other three nucleotide positions induced partial exon skipping. CONCLUSION: Our results underline the potential of understanding the regulation of exonic splicing enhancer sequences and exon skipping therapy for treatment of Duchenne's muscular dystrophy.

Base Sequence↗

PubMed1997

Characterization of the rat insulin-like growth factor I gene promoters and identification of a minimal exon 2 promoter.

Insulin-like growth factor I (IGF-I) promoter activity was characterized in C6, GH3, OVCAR-3, and Chinese hamster ovary (CHO) cells. Maximal exon 1 promoter activity was present in the region extending from -133 to +362 (where +1 is the first transcription start site). Promoter activity was higher in the +75/+362 fragment, which contains exon 1 transcription start sites 3 and 4, than in the -133/+74 fragment, which contains exon 1 transcription start sites 1 and 2. Promoter activity was also observed in constructs containing sequences from -133 to +192, which includes start sites 1, 2, and 3. Inclusion of sequences upstream of -133 inhibited exon 1 proximal promoter activity in a cell type-specific manner. Exon 2 promoter activity was observed in all cell lines with a construct containing 73 bp of 5'-flanking sequence and 44 bp of exon 2. Exon 2 promoter activity was abolished when only 36 bp of 5'-flanking sequence and 44 bp of exon 2 were present, suggesting that an essential minimal promoter element(s) is contained within the -73 to -36 region. A putative CACCC box was observed within this region at -53. Upstream sequence regulated exon 2 promoter activity in a cell type-specific manner. Electrophoretic mobility shift assays revealed a single specifically bound band when the +75/+362 fragment of the exon 1 promoter was used with nuclear extracts from C6 and GH3 cells. Multiple specifically bound bands with slower mobility were observed when the -236/+44 exon 2 promoter fragment was incubated with C6, GH3, CHO, and OVCAR-3 cell nuclear extracts. The exon 1 and exon 2 promoter regions were able to inhibit each other's binding in electrophoretic mobility shift assay using GH3 cell and OVCAR-3 cell nuclear extracts, respectively. Oligonucleotides containing consensus activating protein-1 (AP-1) and AP-3 sequences inhibited exon 1 promoter binding by GH3 cell nuclear extracts. AP-2 and AP-3 sites inhibited exon 2 promoter binding. Our data suggest that the sequence surrounding and including start site 3 in exon 1 functions as a minimal independent promoter. The minimal exon 2 promoter is contained within the 73 bp upstream and 44 bp downstream of the transcription start site cluster. These minimal promoters contain similar and distinct elements that are important for basal transcription. Upstream sequences may contain cell type-specific silencer elements.

Animals↗

PubMed1989

T200 alternate exon use in murine lymphoid cells determined by reverse transcription-polymerase chain reaction.

T200 glycoproteins of lymphoid and myeloid cells exhibit cell lineage-specific structural heterogeneity. Peptide heterogeneity appears to arise from alternate 5'-exon use (Ex-4, 5, and 6), potentially giving rise to eight distinct forms of T200 mRNA containing 0 to 3 of these alternate exons. A method is described for determining the number and identity of the three alternate T200 exons expressed in cells by using the polymerase chain reaction (PCR) and the reverse transcription-polymerase chain reaction (RT-PCR) without prior purification of RNA. Synthetic primers flanking the alternate exon region of T200 were designed to yield products for each possible exon combination having unique size and restriction enzyme sites. PCR amplification of plasmids containing T200 cDNA with none (pLy-5-68) or all three (p70Z/3-3) known alternate exons resulted in the amplification of 186 and 603 bp products, respectively. That amplified products were derived from T200 cDNA was verified by restriction enzyme mapping of each PCR product. T200 cDNA prepared from cell lines utilizing no alternate exons (BW5147) or all three exons (70Z/3.12) were analyzed by RT-PCR and contained amplified products of 186 bp (zero alternate exons) and 603 bp (containing Ex-4+5+6), respectively. RT-PCR of EL4 cells revealed approximately 186 and 330 bp products suggestive of zero and one alternate exon forms. Restriction mapping confirmed that EL4 cells contained a zero-exon form and a one-exon form containing Ex-5. Analysis of the 3B3 pre-B cell line yielded 186, 330, 460, and 603 bp products; restriction mapping revealed T200 mRNA for a zero alternate exon form, two distinct one- and two-exon forms (Ex-4; Ex-5; Ex-4+5; Ex-5+6), and a three-exon form (Ex-4+5+6). Other lymphoid cell lines were heterogeneous in T200 alternate exon use, with distinct patterns distinguishing B and T cells. RT-PCR can facilitate the analysis of variations in T200 alternate exon use among developmentally and functionally distinct lymphoid and myeloid cells.

Animals↗

PubMed1994

The exon 4 poly(A) site of the human calcitonin/CGRP-I pre-mRNA is a weak site in vitro.

The human calcitonin/CGRP-I (CALC-I) pre-mRNA is processed in a tissue-specific alternative way into either calcitonin (CT) or calcitonin gene-related peptide-I (CGRP-I) mRNA. The exons 1 to 3 are common exons. They are spliced to exon 4, which becomes polyadenylated to form CT mRNA, or to exon 5 and the polyadenylated exon 6 to form CGRP-I mRNA. Polyadenylation at exon 4 and splicing of exon 3 to exon 5 are mutually exclusive processing reactions. Only splicing of exon 3 to exon 5 was detected in vitro, with a minigene containing the exon 3 to exon 5 region. No polyadenylation at the exon 4 poly(A) site could be observed. Investigation of the properties of the exon 4 poly(A) site in vitro shows that it is inefficiently used in vitro. Cleavage and polyadenylation of short RNAs containing only the exon 4 poly(A) site is strongly dependent on the 3' length of the RNA. Downstream sequences located within 39 nucleotides from the cleavage site are required for optimal cleavage and polyadenylation. When the exon 4 poly(A) site in the minigene is replaced with the strong adenovirus L3 or rabbit beta-globin poly(A) sites, these sites can be efficiently used in vitro.

Alternative Splicing↗

PubMed2002

Identification of ten exon deleted ERbeta mRNAs in human ovary, breast, uterus and bone tissues: alternate splicing pattern of estrogen receptor beta mRNA is distinct from that of estrogen receptor alpha.

Four different human tissues and breast cancer cell lines were screened to identify exon deletion variant transcripts of estrogen receptor beta (ERbeta) by reverse transcription-polymerase chain reaction using the 'splice targeted primer approach' that amplifies each category of exon deleted variants as a separate gene population. A total of 10 different variant mRNAs that have deletions in various combination of exons were identified by sequence analysis. They were exon 2Delta; exons 2 and 5-6Delta; exon 3Delta; exon 4Delta; exon 5Delta; exons 5 and 2Delta; exon 6Delta; exons 6 and 2Delta; exons 6, 2-3Delta; and exons 5-6Delta. In some cases, deletion of an exon appears to be associated with a mutation of a specific base. Although ERalpha and ERbeta are highly homologous, have identical exon and functional domain organization, exhibit similar ligand-binding profiles and interact with identical DNA response elements, the sequence of exon skipping in ERbeta pre-mRNA appears to be distinct from that of ERalpha mRNA. Furthermore, results described here also suggest that alternate splicing of ERbeta mRNA is tissue specific. The presence of a ERbeta variant profile together with other ER isoforms in a tissue may have functional implications in binding and response to a particular ligand.

Alternative Splicing↗

PubMed1998

Mutations in exon 7 and 8 of p53 as poor prognostic factors in patients with non-small cell lung cancer.

This study was performed to clarify the different effects of each mutant exon of p53 as indicators of a poor prognosis in patients with non-small cell lung cancer (NSCLC). Tumor tissues of 204 patients with NSCLC were analysed; 96 tumors were stage I, 22 stage II, and 86 stage III. DNA was extracted from frozen specimens and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exon 5 to exon 8. Seventy-five patients with NSCLC (36.8%) had mutations in p53 which included 72 cases of missense mutations and three cases of non-missense mutations. The overall survival rate of patients with mutant p53 adenocarcinomas was strikingly worse than that of patients whose tumors had wild-type p53 (35.7% vs 53.8%; P=0.041), but no significant difference in survival was found in the patients with NSCLC and squamous cell carcinoma. Mutations in exon 5 of p53 occurred in 33 cases (16.2%), mutation in exon 6 was detected in only one case (0.5%), mutations in exon 7 in 20 cases (9.8%), and mutations in exon 8 in 18 cases (8.8%). The overall survival rate of patients with mutations in exon 7 was worse than that of patients with wild-type p53 in NSCLCs and adenocarcinomas (42.9% vs 56.0%; P=0.025 and 33.3% vs 53.8%; P=0.048, respectively), whereas the overall survival of patients with mutations in exon 5 was almost the same as that of patients with wild-type p53. In addition, the overall survival rate of patients with mutations in exon 8 was strikingly worse than that of patients with wild-type p53 in NSCLCs, adenocarcinomas and squamous cell carcinomas (22.9% vs 56.0%; P<0.001, 19.0% vs 53.8%; P=0.004 and 33.3% vs 62.5%; P=0.042, respectively). Multivariate analysis with the Cox regression model of patients with NSCLC, adenocarcinoma and squamous cell carcinoma indicated that mutations in exon 8 were best correlated with the overall survival rate, followed by lymph node status (P<0.001, P=0.015 and P=0.006, respectively), and mutations in exon 7 of NSCLC were also revealed to have good correlation, followed by lymph node status and mutations in exon 8 (P=0.031). Mutation of p53 was a poor prognostic factor for adenocarcinoma as described previously. Moreover, mutations in exon 8 were more useful indicators of prognosis not only for adenocarcinoma but also for NSCLC. Worse overall survival of the patients with mutations in exon 8 of p53 was suggested to be associated with codon 273 mutations as well as mutations between codon 280 and 285 included into the H2 alpha helix corresponding to residues 278-286. These results suggested that abnormal conformation of H2 alpha helix might play an important role not only in the loss of normal function but also in the acquisition of tumorigenesis. Investigation of mutations in exon 8, especially codon 273 mutation and mutant H2 alpha helix was considered to be a clinically useful approach for determining the prognosis of patients with NSCLC.

Adenocarcinoma↗

PubMed1995

Structure of the human aggrecan gene: exon-intron organization and association with the protein domains.

The complete exon-intron organization of the human aggrecan gene has been defined, and the exon organization has been compared with the individual domains of the protein core. A yeast artificial chromosome containing the aggrecan gene was selected from the Centre d'Etude du Polymorphisme Humaine yeast artificial chromosome library. A cosmid sulibrary was created from this, and direct sequencing of individual cosmids was used to provide the exon-intron organization. The human aggrecan gene was found to be composed of 19 exons ranging in size from 77 to 4224 bp. Exon 1 is non-coding, whereas exons 2-19 code for a protein core of 2454 amino acids with a calculated mass of 254379 Da. Intron 1 of the gene is at least 13 kb. Overall, the sizes of the 18 introns range from 0.5 to greater than 13 kb. Each intron begins with a GT and ends with an AG, thus obeying the GT/AG rule of splice-junction sequences. The entire coding region is contained in 39.4 kb of the gene. The organization of exons is strongly related to the specific domains of the protein core. The A loop of G1 and the interglobular domain are encoded by exons 3 and 7 respectively. The B and B' loops of G1 are encoded by exons 4-6, and those of G2 are encoded by exons 8-10. These sets of exons, coding for the B and B' loops, are identical in size and organization. This is supported by the intron classes associated with these exons. Exon 11 codes for the 5' half of the keratan sulphate-rich region, and exon 12 codes for the 3' half of the keratan sulphate-rich region as well as the entire chondroitin sulphate-rich region. G3 is encoded by exons 13-18, including the alternatively spliced epidermal growth factor-like and complement regulatory protein-like domains. The correspondence between the exon organization and the protein domains argues strongly for modular assembly of the aggrecan gene.

Aggrecans↗

PubMed2003

c-kit gene mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors.

BACKGROUND AND AIM: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the human gut. They frequently have gain-of-function mutations of the c-kit gene, which encodes a receptor, tyrosine kinase. The mutations were found at exon 11 in most cases, and either at exon 9 or at exon 13 in rare cases. Recently, we found a family with multiple GIST and a gain-of-function mutation at exon 17. The family was the first reported GIST case with c-kit gene mutation at exon 17 including sporadic GIST. Although we previously reported that the c-kit gene mutation at exon 17 was not detected in 124 sporadic GIST by single-strand conformation polymorphism (SSCP) analysis, the mutation at exon 17 observed in the familial GIST was detectable by the use of direct sequencing but not by our SSCP method. In the present study, we examined the mutations at exon 17 and exon 13 by using direct sequencing. METHODS: Genomic DNA was extracted from formalin-fixed, paraffin-embedded GIST tissues. We could obtain 143 sporadic GIST cases appropriate for DNA analysis at exon 17 and 141 at exon 13. Exons 17 and 13 were amplified by using polymerase chain reaction and direct sequencing was conducted. RESULTS: No mutation was found at exon 17, and only one case with the mutation at exon 13 was observed. The GIST with the mutation at exon 13 was large and showed frequent mitosis, and the patient died of the recurrent GIST 3 years after the first operation. CONCLUSION: The mutation at exons 17 or 13 was considered to be very rare in sporadic GIST.

Aged↗

PubMed1999

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.

Alternative Splicing↗

PubMed2005

Tau exons 2 and 10, which are misregulated in neurodegenerative diseases, are partly regulated by silencers which bind a SRp30c.SRp55 complex that either recruits or antagonizes htra2beta1.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 2 modulates the tau N-terminal domain, which interacts with the axonal membrane. Exon 10 codes for a microtubule binding domain, increasing the affinity of tau for microtubules. Both exons are excluded from fetal brain, but their default behavior is inclusion, suggesting that silencers are involved in their regulation. Exon 2 is significantly reduced in myotonic dystrophy type 1, whose symptoms include dementia. Mutations that affect exon 10 splicing cause frontotemporal dementia (FTDP). In this study, we investigated three regulators of exon 2 and 10 splicing: serine/arginine-rich (SR) proteins SRp55, SRp30c, and htra2beta1. The first two inhibit both exons; htra2beta1 inhibits exon 2 but activates exon 10. By deletion analysis, we identified splicing silencers located at the 5' end of each exon. Furthermore, we demonstrated that SRp30c and SRp55 bind to both silencers and to each other. In exon 2, htra2beta1 binds to the inhibitory heterodimer through its RS1 domain but not to exon 2, whereas in exon 10 the heterodimer may sterically interfere with htra2beta1 binding to a purine-rich enhancer (defined by FTDP mutation E10-Delta5 = Delta280K) directly downstream of the silencer. Increased exon 10 inclusion in FTDP mutant ENH (N279K) may arise from abolishing SRp30c binding. Also, htra2beta3, a naturally occurring variant of htra2beta1, no longer inhibits exon 2 splicing but can partially rescue splicing of exon 10 in FTDP mutation E10-Delta5. This work provides interesting insights into the splicing regulation of the tau gene.

Animals↗

PubMed1991

Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.

Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene.

AMP Deaminase↗

PubMed1994

Identification of exon sequences involved in splice site selection.

The involvement of exon sequences in splice site selection was studied in vivo in HeLa cells transfected with a series of model three exon-two intron pre-mRNAs which differed only in the sequence of their internal exons. When the majority of the human globin-derived 175-nucleotide internal exon (DUP175) was replaced with a sequence from the yeast URA3 gene (DUP184), the splicing pathway changed from complete inclusion of the internal exon in DUP175 to its predominant skipping in the DUP184 construct. Skipping of the exon was reversed by increasing the strength of its flanking splicing elements indicating that exon sequences exert their effect only in the presence of relatively weak splicing signals. A series of block mutations in the internal exon of DUP184 showed that a stretch of 6 cytidine nucleotides increased the inclusion of the DUP184 internal exon about 7-fold. Mutations generating purine-rich sequences (AAG and GAAG) at the 3' end of the exon led to complete exon inclusion while stepwise insertion of sequences from the internal exon of DUP175 into the DUP184 background increased exon inclusion 5-fold. Combination of the stretch of cytidines with sequences derived from DUP175 exon resulted in complete exon inclusion indicating that diverse signals within exons may cooperate with each other in affecting splice site selection.

Base Sequence↗

PubMed1993

A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III.

Four distinct FGF receptors were cloned and characterized and it was demonstrated that the ligand binding site of FGF receptors is confined to the extracellular immunoglobulin-like (Ig)-domain 2 and 3. The Ig-domain 3 is encoded by two separate exons: exon IIIa encodes the N-terminal half, and the C-terminal half is encoded by either exon IIIb or IIIc in FGFR1 and FGFR2, whereas FGFR4 is devoid of exon IIIb. Alternative usage of exons IIIb and IIIc determine the ligand binding specificity of the receptor. To analyze the arrangement of these exons in FGFR3 we cloned the genomic sequence between exon IIIa and IIIc of FGFR3 and identified an alternative exon, corresponding to exon IIIb of the FGFR1 and FGFR2. The sequence of this exon shows Ig-domain hallmarks, 44% identity with exon IIIb of other FGF receptors and 36% identity with exon IIIc of FGFR3. Using this exon as a probe for mouse RNA as well as PCR analysis, demonstrated that exon IIIb encodes an authentic form of FGFR3 that is expressed in mouse embryo, mouse skin and mouse epidermal keratinocytes. The results demonstrate that the presence of alternative exons for Ig-domain 3 is a general phenomena in FGFR1, 2 and 3, and represents a novel genetic mechanism for the generation of receptor diversity.

Amino Acid Sequence↗

PubMed2005

Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion.

AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B. delaTorre, P. Taylor, Knockout mice with deletions of alternatively spliced exons of Acetylcholinesterase, in: N.C. Inestrosa, E.O. Campus (Eds.), VII International Meeting on Cholinesterases, Pucon-Chile Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects. P. Universidad Catholica de Chile-FONDAP Biomedicina, 2004, pp. 43-48; R.Y.Y. Chan, C. Boudreau-Larivière, L.A. Angus, F. Mankal, B.J. Jasmin, An intronic enhancer containing an N-box motif is required for synapse- and tissue-specific expression of the acetylcholinesterase gene in skeletal muscle fibers. Proc. Natl. Acad. Sci. USA 96 (1999) 4627-4632], is also presented. The intronic region was floxed and then deleted by mating with Ella-cre transgenic mice. The deletion of this region produced a dramatic phenotype; a mouse with near normal AChE expression in brain and other CNS tissues, but no AChE expression in muscle. Phenotype and AChE tissue activities are compared with the total AChE knockout mouse [W. Xie, J.A. Chatonnet, P.J. Wilder, A. Rizzino, R.D. McComb, P. Taylor, S.H. Hinrichs, O. Lockridge, Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase. J. Pharmacol. Exp. Ther. 293 (3) (2000) 896-902].

Acetylcholinesterase↗

PubMed1994

Large exon size does not limit splicing in vivo.

Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

Animals↗

PubMed2000

An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein.

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for "intronic splicing activator and repressor") which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. We have now identified an intronic element upstream of exon IIIb that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms were equally capable of mediating this effect. Our results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb.

Adenocarcinoma↗

PubMed2006

The effects of multiple features of alternatively spliced exons on the K(A)/K(S) ratio test.

BACKGROUND: The evolution of alternatively spliced exons (ASEs) is of primary interest because these exons are suggested to be a major source of functional diversity of proteins. Many exon features have been suggested to affect the evolution of ASEs. However, previous studies have relied on the KA/KS ratio test without taking into consideration information sufficiency (i.e., exon length > 75 bp, cross-species divergence > 5%) of the studied exons, leading to potentially biased interpretations. Furthermore, which exon feature dominates the results of the KA/KS ratio test and whether multiple exon features have additive effects have remained unexplored. RESULTS: In this study, we collect two different datasets for analysis - the ASE dataset (which includes lineage-specific ASEs and conserved ASEs) and the ACE dataset (which includes only conserved ASEs). We first show that information sufficiency can significantly affect the interpretation of relationship between exons features and the KA/KS ratio test results. After discarding exons with insufficient information, we use a Boolean method to analyze the relationship between test results and four exon features (namely length, protein domain overlapping, inclusion level, and exonic splicing enhancer (ESE) frequency) for the ASE dataset. We demonstrate that length and protein domain overlapping are dominant factors, and they have similar impacts on test results of ASEs. In addition, despite the weak impacts of inclusion level and ESE motif frequency when considered individually, combination of these two factors still have minor additive effects on test results. However, the ACE dataset shows a slightly different result in that inclusion level has a marginally significant effect on test results. Lineage-specific ASEs may have contributed to the difference. Overall, in both ASEs and ACEs, protein domain overlapping is the most dominant exon feature while ESE frequency is the weakest one in affecting test results. CONCLUSION: The proposed method can easily find additive effects of individual or multiple factors on the KA/KS ratio test results of exons. Therefore, the system can analyze complex conditions in evolution where multiple features are involved. More factors can also be added into the system to extend the scope of evolutionary analysis of exons. In addition, our method may be useful when orthologous exons can not be found for the KA/KS ratio test.

Alternative Splicing↗

PubMed1988

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene.

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.

Antigens, Differentiation↗