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PubMed1992

Alternative splicing in the human lck gene leads to the deletion of exon 1' and results in a new type II lck transcript.

In this study we describe a new lck mRNA transcribed from the type II promoter, resulting from alternative splicing, in the deletion of exon 1' which comprises the AUG initiation codon. In this type IIB lck transcript, as a consequence of exon 1' deletion, a new AUG codon in exon 1, located 35 nt upstream from the regular initiation codon in exon 1', and normally out of frame, is now in frame with exon 2 and the following lck coding sequence. In this type IIB lck transcript, 10 residues encoded by exon 1 from the new AUG codon replace the first 35 residues encoded by exon 1'. Strikingly, in this new N-terminal amino acid sequence, a glycine residue, needed for myristylation and anchorage in the plasma membrane, is present at position 2. This alternative splicing has been observed in T-cell lines, normal mature T cells and in peripheral blood lymphocytes from different leukemic patients. Different ratios of regular type IIA lck mRNA (with exon 1') to spliced type IIB lck mRNA (without exon 1') have been observed depending on the patient.

Alternative Splicing↗

PubMed1989

Lymphomagenesis in Emu-myc transgenic mice does not require transgene rearrangement or mutation of myc exon 1.

In most human Burkitt's lymphomas, translocation of the myc oncogene to an immunoglobulin locus is associated with loss of myc exon 1 or with mutations near its 3' border, a region where myc transcription is attenuated and translation of a larger myc polypeptide initiates. Emu-myc transgenic mice, which bear the three myc exons coupled to an immunoglobulin enhancer, provide a model for the development of such lymphomas, because their lymphomagenesis appears to require events other than expression of the transgene. To determine whether myc rearrangement or exon 1 mutation is a necessary tumorigenic event, we examined the transgene structure and myc exon 1 sequences in Emu-myc B lymphoid tumours. Southern blots revealed no transgene rearrangements in 20 of the lymphomas, and only two tumours showed amplification (2 to 5-fold). To search for exon 1 alterations, the exon 1 mRNA region was amplified from five tumours by polymerase chain reaction and sequenced, but no mutations were found. Hence, neither excision nor mutation of exon 1 is necessary to render myc tumorigenic. The sequence analysis across the exon 1-exon 2 boundary unexpectedly revealed an ambiguity in myc splicing that predicts a variant form of the larger myc polypeptide lacking a single amino acid residue.

Amino Acid Sequence↗

PubMed1987

Exon structure of a mannose-binding protein gene reflects its evolutionary relationship to the asialoglycoprotein receptor and nonfibrillar collagens.

Cloned cDNAs encoding mannose-binding proteins isolated from rat liver have been used to isolate one of the genes encoding this group of proteins. This gene, which encodes the minor form of binding protein (designated MBP-A), has been characterized by sequence analysis. The protein-coding portion of the mRNA for the MBP-A is encoded by four exons separated by three introns. The NH2-terminal, collagen-like portion of the protein is encoded by the first two exons. These exons resemble the exons found in the genes for nonfibrillar collagens in that the intron which divides them is inserted between the first two bases of a glycine codon and the exons do not have the 54- or 108-base pair lengths characteristic of fibrillar collagen genes. The carbohydrate-binding portion of MBP-A is encoded by the remaining two exons. This portion of the protein is homologous to the carbohydrate-recognition domain of the hepatic asialoglycoprotein receptor, which is encoded by four exons. It appears that the three COOH-terminal exons of the asialoglycoprotein receptor gene have been fused into a single exon in the MBP-A gene. The organization of the MBP-A gene is very similar to the arrangement of the gene encoding the highly homologous pulmonary surfactant apoprotein, although one of the intron positions is shifted by a single amino acid. The 3' end of a mannose-binding protein pseudogene has also been characterized.

Amino Acid Sequence↗

PubMed1995

An aberrant splicing using a 3' cryptic splice site within the CH1 exon induces truncated mu-chain production.

AT8--1-12--5-1, an Abelson virus-transformed immature B-cell line, produced truncated mu-chains. Sequencing analysis of the mu-expressed allele revealed that the variable region was an out-of-frame VH7183-DSP2-JH3 complex. Two cDNA clones (5-1 cDNA1 and 5-1 cDNA2) derived from the transcripts of the mu-expressed allele were cloned and sequenced. Sequencing analysis of 5-1 cDNA1 revealed that the VH7183-DSP2-JH3 sequence jointed to the CH1 exon at 136 bp, 3' from the 5' end of the CH1 exon, resulting in the change of the reading frame from out-of-frame to in-frame. On the other hand, sequencing analysis of 5-1 cDNA2, which appeared to have derived from intron-containing premature mRNA, revealed that the J-C intron sequence joined to the CH1 exon at 110 bp 3' from the 5' end of the CH1 exon, indicating the deletion of 109 bp including the 3' splice site of the CH1 exon. These results demonstrate that the deletion of the authentic 3' splice site of the CH1 exon induced activation of the cryptic splice site within the CH1 exon. This was followed by splicing of the variable region to the CH1 exon at the cryptic splice site at 136 bp 3' from the 5' end of the CH1 exon, resulting in the change of the reading frame from out-of-frame to in-frame, followed by the truncated mu-chain production.

Alleles↗

PubMed1995

Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.

Amino Acid Sequence↗

PubMed1994

Distribution of alternatively spliced chicken c-myb exon 9A among hematopoietic tissues.

The role of the c-myb proto-oncogene in cellular differentiation may be regulated in part by alternative splicing of its mRNAs. Previously, two forms of alternative splicing of the chicken c-myb gene between exons 9 and 10 were described: one form utilizes the entire 360 base pair (bp) exon 9A while a second form utilizes exon 9A' which consists of the 3' 150 bp of exon 9A. In this study the distribution among chicken hematopoietic tissues of these two forms of alternative splicing was determined by Northern blot analysis using a probe specific for exon 9A. RNA species of 4.2 kilobases (kb) and 4.4 kb which contain exon 9A' or exon 9A, respectively, were detected in each tissue tested. Quantitative analysis of the major 4.0 kb c-myb species and the c-myb species containing exon 9A and exon 9A' revealed that cells from yolk sac contained both the highest absolute and the highest relative levels of alternatively spliced c-myb mRNA, presumably because of the preponderance of immature erythroid cells in these preparations.

Alternative Splicing↗

PubMed1994

Demonstration of promoter activity and alternative splicing in the region 5' to exon 1 of the APC gene.

Screening of fetal brain and fetal retina complementary DNA (cDNA) libraries and exon-connection experiments using brain cDNA have identified three exons 5' to exon 1 of the adenomatous polyposis coli gene. The exons are termed (from 5'-3') 0.3, 0.1, and 0.2; exons 0.1 and 0.2 are contiguous genomically. Library screening revealed alternatively spliced cDNAs containing the following combinations of 5'-exons: 0.3 + 1 + 2, 0.3 + 2, 0.1 + 0.2 + 1 + 2, and 0.1 + 1 + 2. Exon-connection experiments also identified these four forms in mRNAs from tissues and cultured cell lines, along with two additional forms, 0.1 + 0.2 + 2 and 0.1 + 2. The multiple splice forms may lead to proteins of differing activity; for example, products derived from cDNAs without exon 1 will lack most of a heptad-repeat domain that supports formation of homodimers. No mRNA species combining 0.3 with either 0.1 or 0.2 were identified. The existence of two apparently separate 5'-ends of APC suggests the possibility of two independent promoters. The genomic sequence adjacent to exon 0.3 confers promotor activity when cloned in a chloramphenicol acetyltransferase expression vector and transfected into a colon cancer cell line.

Alternative Splicing↗

PubMed1996

FGFR2 exon IIIa and IIIc mutations in Crouzon, Jackson-Weiss, and Pfeiffer syndromes: evidence for missense changes, insertions, and a deletion due to alternative RNA splicing.

Fibroblast growth factor receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgIII) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in 17 unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders.

Acrocephalosyndactylia↗

PubMed1996

Exon circularization in mammalian nuclear extracts.

Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

Base Sequence↗

PubMed1996

A case of Becker muscular dystrophy resulting from the skipping of four contiguous exons (71-74) of the dystrophin gene during mRNA maturation.

The mutations in one-third of both Duchenne and Becker muscular dystrophy patients remain unknown because they do not involve gross rearrangements of the dystrophin gene. Here we report the first example of multiple exon skipping during the splicing of dystrophin mRNA precursor encoded by an apparently normal dystrophin gene. A 9-year-old Japanese boy exhibiting excessive fatigue and high serum creatine kinase activity was examined for dystrophinopathy. An immunohistochemical study of muscle tissue biopsy disclosed faint and discontinuous staining of the N-terminal and rod domains of dystrophin but no staining at all of the C-terminal domain of dystrophin. The dystrophin transcript from muscle tissue was analyzed by the reverse transcriptase polymerase chain reaction. An amplified product encompassing exons 67-79 of dystrophin cDNA was found to be smaller than that of the wild-type product. Sequence analysis of this fragment showed that the 3' end of exon 70 was directly connected to the 5' end of exon 75 and, thus, that exons 71-74 were completely absent. As a result, a truncated dystrophin protein lacking 110 amino acids from the C-terminal domain should result from translation of this truncated mRNA, and the patient was diagnosed as having Becker muscular dystrophy at the molecular level. Genomic DNA was analyzed to identify the cause of the disappearance of these exons. Every exon-encompassing region could be amplified from genomic DNA, indicating that the dystrophin gene is intact. Furthermore, sequencing of these amplified products did not disclose any particular nucleotide change that could be responsible for the multiple exon skipping observed. Considering that exons 71-74 are spliced out alternatively in some tissue-specific isoforms, to suppose that the alternative splicing machinery is present in the muscle tissue of the index case and that it is activated by an undetermined mechanism is reasonable. These results illustrate a novel genetic anomaly that results in dystrophinopathy.

Child↗

PubMed1997

The exon 46-encoded sequence is essential for stability of human erythroid alpha-spectrin and heterodimer formation.

Human erythroid alpha-spectrin alleles responsible for hereditary elliptocytosis (alphaHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism alphaLELY (LELY: Low Expression LYon) occurs in trans. The alphaLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA --> GTA, Leu --> Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the alpha-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of alpha-chains from the alphaLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif alpha subunit peptides containing the dimer initiation region. The two forms of alpha spectrin produced by alternative mRNA splicing of the alphaLELY allele were represented by alpha18-21(1857), a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and alpha18-21(1857-Delta46), a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with alpha18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the alphaIV and alphaV domains that was the characteristic feature initially used to identify the alphaLELY (SpalphaV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the alpha21 motif, because this region of the alpha18-21(1857-Delta46) peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an alphaLELY homozygote and the two alphaLELY recombinant peptides strongly suggests that little, if any, of the 50% of the alpha chains from the alphaLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant alphaLELY peptides, the inability of detectable amounts of exon 46(-) alpha chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.

Alleles↗

PubMed1997

The IGHG3 gene shows a structural polymorphism characterized by different hinge lengths: sequence of a new 2-exon hinge gene.

Four of the five human IGHG genes (G1, GP, G2, and G4) display a hinge region consisting of a unique exon. In contrast, IGHG3 exhibits a different structure in which the hinge is constituted by four or, less frequently, three exons. We report here the nucleotide sequence of a new 2-exon hinge G3 gene found in a Mandenka individual from Eastern Senegal. A comparison of this sequence with that of 4-exon and 3-exon hinge G3 genes suggests that the 3-exon and 2-exon hinge forms arose independently by deletion events in a 4-exon hinge gene.

B-Lymphocytes↗

PubMed1999

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA.

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell-specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte- and mesenchymal cell-derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type-specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included.

Alternative Splicing↗

PubMed1997

Constitutive APC exon 14 skipping in early-onset familial adenomatous polyposis reveals a dramatic quantitative distortion of APC gene-specific isoforms.

Adenomatous polyposis coli (APC) gene transcripts skipping exon 14 in combination with the alternatively spliced exons 9 and 10A contribute to the heterogeneity of physiological APC mRNA isoforms. Here we report on a novel genotype-phenotype correlation in familial adenomatous polyposis (FAP) with early onset of disease and malignancy due to an APC exon 14 splice defect. Compared to controls, two affected individuals of a FAP kindred presented with a significantly distorted APC mRNA isoform pattern in B lymphocytes. As a result of an A-->G transition in the canonical AG-splice acceptor dinucleotide of exon 14, expression levels of all APC mRNA isoforms without exon 14 were dramatically increased and those with exon 14 were simultaneously decreased. Skipping of exon 14 is a physiological event also seen in nonmalignant cells, which results in a frameshift to produce low-molecular-weight APC proteins. Western blot analysis of the patients' lymphoblastoid B cells revealed the identification of intracellularly stable APC protein isoforms with an Mr of 55-67 kDa and, thus, the first demonstration of APC proteins encoded by exon 14-skipped transcripts. We postulate that the quantitatively imbalanced expression of these physiological APC light chains represents a novel pathogenetic mechanism associated with predisposition to FAP.

Adenomatous Polyposis Coli↗

PubMed2007

Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy.

Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients.

Adolescent↗

PubMed1987

Primary structure of the gene for the murine Ia antigen-associated invariant chains (Ii). An alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin.

The gene for murine Ia-associated invariant (Ii) chains (Ii31 and Ii41) was characterized by sequence analysis. The gene extends over approximately 9 kb and is organized in nine exons. Exon 1 encodes the 5' untranslated region and the cytoplasmic segment, exon 2 the membrane spanning segment and adjacent amino acids and exons 3-8 the extracytoplasmic portion of Ii31. Putative promoter sequences were found upstream of the start of the coding sequence. Between exons 6 and 7 an additional, alternatively spliced exon 6b has been identified. This exon is spliced into the mRNA coding for the Ii-related Ii41 protein. Exon 6b encodes a cysteine-rich domain of 64 amino acids. It shows a remarkably high homology to the repetitive elements in thyroglobulin, a precursor for thyroid hormone. Based on this homology, it is suggested that this domain (TgR) in Tg and in Ii41 may play a role either in hormone formation or as a carrier in the transport of molecules (thyroid hormone or processed antigen respectively) between intracellular compartments.

Amino Acid Sequence↗

PubMed1987

Self-splicing of a group II intron in yeast mitochondria: dependence on 5' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.

Base Sequence↗

PubMed2004

Computer-identified nuclear localization signal in exon 1A of the transporter DMT1 is essentially ineffective in nuclear targeting.

Divalent metal transporter 1 (DMT1; also called DCT1, Nramp2, or SLC11A2) has multiple isoforms that localize differently in many cell types. DMT1 +IRE species (encoded by mRNA with an iron-responsive element) are limited to the plasma membrane and cytosolic vesicles. In neural cells, -IRE isoforms of DMT1 (encoded by mRNA lacking an IRE) localize to the nucleus, plasma membrane, and cytosolic vesicles. In considering nuclear compartmentalization of -IRE isoforms, we hypothesized that the newly identified exon 1A in the N-terminus of this transporter might contain a nuclear localization signal. DNA constructs starting with exon 1A and ending with exons encoding alternative isoforms were made and transiently transfected into HEK293T and PC12 cells as well as rat sympathetic neurons. None of the constructs appeared in the nucleus despite the presence of exon 1A. Antibody specific for exon 1A was also used in both immunostaining and Western blots to investigate localization of exon 1A expressed both endogenously and ectopically in cells. Again, nuclear localization of DMT1 containing exon 1A was not observed. Our data suggest that exon 1A is neither sufficient nor necessary for DMT1 to appear in the nucleus.

Animals↗