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PubMed1999

A single base mutation in the 5' splice site of intron 7 of the lck gene is responsible for the deletion of exon 7 in lck mRNA of the JCaM1 cell line.

The failure of signal transduction in the JCaM1 cell line was associated with the presence of an abnormal lck mRNA deleted of the exon 7 encoding for an inactive p56lck kinase. Our study of the lck mRNA from various T cell lines and from peripheral blood lymphocytes of healthy donors has revealed the presence of both complete and exon 7-deleted lck transcripts. Thus the exon 7-deleted lck transcript initially described in the JCaM1 mutant cell line, arises from an alternative splicing event occurring in each cells expressing the lck gene. Genomic DNA sequencing of the lck exons 6-8 portion from both the mutant JCaM1 and its parental Jurkat cell lines revealed as the only difference, the presence of a A to G mutation within the 5' splice site of intron 7 in the JCaM1 cell line DNA. To demonstrate the role of this point mutation in the lck pre-mRNA maturation, COS cells were transfected by lck minigenes from the Jurkat and JCaM1 cell lines. In COS cells transfected with minigene from the Jurkat cell line both lck transcripts (with and without exon 7) were observed whereas only the exon 7-spliced lck transcript was observed in COS cells transfected with minigene from the JCaM1 cell line. Thus the mutation is per se responsible for the deletion of exon 7 and the absence of complete lck mRNA in the JCaM1 cell line. Presence of a restriction site (HphI) in the 5' splice site of lck intron 7 from Jurkat DNA allowed to confirm the presence of the mutation on both alleles in the JCaM1 cell line.

Animals↗

PubMed2001

A melanoma-associated germline mutation in exon 1beta inactivates p14ARF.

The INK4a/ARF locus encodes the cyclin dependent kinase inhibitor, p16(INK4a) and the p53 activator, p14ARF. These two proteins have an independent first exon (exon 1alpha and exon 1beta, respectively) but share exons 2 and 3 and are translated in different reading frames. Germline mutations in this locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Although most of these mutations specifically inactivate p16(INK4a), more than 40% of the INK4a/ARF alterations located in exon 2, affect both p16(INK4a) and p14ARF. We now report a 16 base pair exon 1beta germline insertion specifically altering p14ARF, but not p16(INK4a), in an individual with multiple primary melanomas. This mutant p14ARF, 60ins16, was restricted to the cytoplasm, did not stabilize p53 and was unable to arrest the growth of a p53 expressing melanoma cell line. This is the first example of an exon 1beta mutation that inactivates p14ARF, and thus implicates a role for this tumour suppressor in melanoma predisposition.

Adult↗

PubMed1998

Characterization of exon skipping mutants of the COP1 gene from Arabidopsis.

The removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1-1 was a G-->A change 4 nt upstream from the 3' splice site of intron 5, while the mutation in cop1-2 was a G-->A at the first nucleotide of intron 6, abolishing the conserved G within the 5' splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1-8 was G-->A in the final nucleotide of intron 10 abolishing the conserved G within the 3' splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.

Arabidopsis↗

PubMed1998

Splicing of a regulated exon reveals additional complexity in the axonal microtubule-associated protein tau.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression pattern and splicing regulation had not been previously analyzed in the human. The expression profile of exon 6 is completely different from that of the better-analyzed exons 2, 3, 4A, and 10, implying the utilization of distinct regulatory factors. The default splicing behavior of the exon had demonstrated the existence of what were initially considered cryptic splice sites. However, analysis of the expression pattern of exon 6 suggests that these splice sites are utilized in certain human tissues and, if translated, would result in a radically altered tau protein. Lastly, expression of exon 6 minigene constructs in cells indicates that its flanking exons are involved in its inclusion and in the modulation of the ratio of its variants.

Amino Acid Sequence↗

PubMed2002

Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut.

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.

5' Untranslated Regions↗

PubMedDate not supplied

Duplication of exon 3 in the glycophorin C gene gives rise to the Lsa blood group antigen.

BACKGROUND: The Gerbich-related Lsa blood group antigen (Ge6) resides on the higher-molecular-weight forms of glycophorin C (GPC) and glycophorin D (GPD). Southern blot analysis has previously revealed an additional GPC exon 3 insert in the genomic DNA from an Ls(a+) individual. STUDY DESIGN AND METHODS: To confirm the duplication of exon 3 in the GPC mRNA, total RNA prepared from the Epstein-Barr virus-transformed lymphocytes of an Ls(a+) individual was used in the synthesis of first-strand cDNA. The first-strand cDNA served as a template for the amplification of GPC-related DNA by polymerase chain reaction. After subcloning, the polymerase chain reaction cDNA was sequenced with a kit. Hemagglutination inhibition of anti-Lsa sera with synthetic peptides was performed to identify the location of Lsa on the GPC.Lsa protein. RESULTS: Sequencing the GPC.Lsa cDNA showed an insert of 84 nucleotides, which corresponds to the entire sequence of exon 3 in the GPC gene (GYPC). Since the exon 3-duplicated exon 3 boundary of GYPC.Lsa encodes a novel amino acid sequence on GPC.Lsa and GPD.Lsa, synthetic peptides consisting of the amino acids flanking this junction were used to determine the amino acid sequence that is essential for expression of Lsa. The peptide DIVVIA/EPDPG was noninhibitory, while peptides with the sequence TPTIMDIVVIA/EPDPG or SPSVLDIVVIA/EPDPG inhibited anti-Lsa reactivity. CONCLUSION: Lsa is located within the sequence of amino acids encoded by the nucleotides at the exon 3-duplicated exon 3 boundary, and the conformation of this peptide sequence is important for recognition of Lsa by human anti-Lsa.

Amino Acid Sequence↗

PubMedDate not supplied

The insulin receptor isoform exon 11- (IR-A) in cancer and other diseases: a review.

The insulin receptor plays a vital role in mediating the actions of insulin. These include metabolic and mitogenic effects. This review will focus on the role of the insulin receptor isoforms in normal development and the pathogenesis of certain cancers and type 2 diabetes. There are two insulin receptor isoforms arising from the alternative splicing of exon 11 resulting in either the exon 11+ (IR-B) isoform (including 12 amino acids encoded by exon 11) or the exon 11- (IR-A) isoform. The isoforms have different affinities for insulin, IGF-II and IGF-I with the exon 11- isoform binding both insulin and IGF-II with high affinities. Interestingly, differential expression of the insulin receptor isoforms has been demonstrated in disease. Several cancer cell types that also overexpress IGF-II preferentially express the exon 11- isoform. Activation of the exon 11- insulin receptor by IGF-II and insulin results in mitogenic effects and a potentiation of the cancer phenotype. Also hyperinsulinemia has been associated with increased risk of cancer. Differential expression of the insulin receptor isoforms has also been demonstrated in type 2 diabetes although there is some discrepancy in the literature as to which isoform is expressed.

Amino Acid Sequence↗

PubMed2004

Single-nucleotide promoter polymorphism alters transcription of neuronal nitric oxide synthase exon 1c in infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis (IHPS), characterized by enlarged pyloric musculature and gastric-outlet obstruction, is associated with altered expression of neuronal nitric oxide synthase (nNOS). Here we have studied molecular mechanisms by which nNOS gene expression is altered in pyloric tissues of 16 infants with IHPS and 9 controls. A significant decreased expression of total nNOS mRNA was found by quantitative RT-PCR in IHPS after normalization against GAPDH, which predominantly affected exon 1c with a reduction of 88% compared with controls (P < 0.001). After normalization against the neuronal-specific gene PGP9.5, expression of exon 1c was still decreased (P < 0.001), whereas expression of exon 1f was increased significantly (P = 0.001), indicating a compensatory up-regulation of this nNOS mRNA variant. DNA samples of 16 IHPS patients and 81 controls were analyzed for nNOS exon 1c promoter mutations and single-nucleotide polymorphism (SNP). Sequencing of the 5'-flanking region of exon 1c revealed mutations in 3 of 16 IHPS tissues, whereas 81 controls showed the wild-type sequence exclusively. Carriers of the A allele of a previously uncharacterized nNOS exon 1c promoter SNP (-84G --> A) had increased risk for development of IHPS (odds ratio, 8.0; 95% confidence interval, 2.5-25.6). Reporter gene assays revealed an unchanged promoter activity for mutations but a approximately 30% decrease for the -84A SNP (P < 0.001). In summary, our findings indicate that genetic alterations in the nNOS exon 1c regulatory region influence expression of the nNOS gene and may contribute to the pathogenesis of IHPS.

Base Sequence↗

PubMed1991

Marked increases of two kinds of two-exon-skipped albumin mRNAs with aging and their further increase by treatment with 3'-methyl-4-dimethylaminoazobenzene in Nagase analbuminemic rats.

Nagase analbuminemic rats (NARs) have a 7-base-pair deletion at the 5' splice site of the HI intron of the albumin gene. The level of immunohistochemically albumin-positive hepatocytes is about 1 per 10(5) cells in neonatal NARs, increases with age, and further increases with chronic oral treatment with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). The mechanisms involved in the increase in albumin-positive hepatocytes during aging of NARs and their treatment with 3'-MeDAB were analyzed. NARs were found to have four species of albumin mRNA: intact mRNA and those lacking the regions corresponding to exon H, exon G-H, and exon H-I. In 4-week-old NARs, the level of intact albumin mRNA was about 1/4000 of that in normal rats and mRNA lacking the exon H sequence was the major species. In aged and 3'-MeDAB-treated aged NARs, all four species of mRNA increased and the relative proportion of mRNAs lacking two exon sequences to mRNAs lacking one exon sequence was greatly increased, suggesting that aging is associated with changes of the splicing pattern and that 3'-MeDAB treatment enhanced these changes. In aged NARs and 3'-MeDAB-treated aged NARs, there was an increase in the amount of aberrant 60-kDa albumin. The 60-kDa protein could be a translation product of mRNAs lacking two exons, the amount of which increases in aged NARs and 3'-MeDAB-treated NARs.

Aging↗

PubMed1992

Establishment of a highly sensitive and specific exon-trapping system.

We have established a highly sensitive and specific exon-trapping system (SETS) with a specific plasmid vector in which an exon in a given DNA segment is identified by its ability to remain as a mature mRNA after splicing. The SETS provides us with the isolation of possible exons rapidly and easily from DNA fragments in chromosomal regions of more than 300 kilobase pairs. Genomic DNA fragments were partially digested and subsequently cloned into plasmid pMHC2, an exon-trapping vector we have constructed. These constructs were transfected into COS-7 cells, and consequent RNA transcripts were spliced in the cells. The resulting mature mRNA was harvested and amplified by using reverse transcription-PCR. Possible exons can be recognized by the sizes of PCR products and cloned into a plasmid vector. The SETS provides a direct means of cloning exons from genomic DNA of more than 300 kilobase pairs within a short period of time. Using this system, we have screened 300-kilobase-pair genomic DNA segments derived from human chromosome 11q13. Human chromosome 11q13 may contain genes responsible for human cancers, because DNA amplification is observed in several malignant tumors. We have successfully identified exon 2 of the HST1 gene and additional transcribed sequences.

Base Sequence↗

PubMed1998

SR proteins are sufficient for exon bridging across an intron.

We have developed a defined system to characterize the role of SR proteins and exonic enhancers in directly promoting splice-site interactions across an intron. Using RNA affinity chromatography, we find that SR proteins alone are sufficient to promote the specific association of the enhancer-containing exon 5 with the adjoining exon 6 from avian cardiac troponin-T. Direct visualization of this exon/exon association by electron spectroscopic imaging shows it to be highly specific. Furthermore, using in vivo characterized mutants of exon 5, we also show that this exon/exon association depends on the splicing enhancer within exon 5. These results suggest a model by which SR proteins may function through exonic enhancers to directly promote exon bridging.

Alternative Splicing↗

PubMed1999

An exon that prevents transport of a mature mRNA.

In Caenorhabditis elegans, pre-mRNA for the essential splicing factor U2AF65 sometimes is spliced to produce an RNA that includes an extra 216-bp internal exon, exon 3. Inclusion of exon 3 inserts an in-frame stop codon, yet this RNA is not subject to SMG-mediated RNA surveillance. To test whether exon 3 causes RNA to remain nuclear and thereby escape decay, we inserted it into the 3' untranslated region of a gfp reporter gene. Although exon 3 did not affect accumulation or processing of the mRNA, it dramatically suppressed expression of green fluorescent protein (GFP). We showed by in situ hybridization that exon 3-containing gfp RNA is retained in the nucleus. Intriguingly, exon 3 contains 10 matches to the 8-bp 3' splice-site consensus. We hypothesized that U2AF might recognize this octamer and thereby prevent export. This idea is supported by RNA interference experiments in which reduced levels of U2AF resulted in a small burst of gfp expression.

Animals↗

PubMed1995

The precursors of the bee venom constituents apamin and MCD peptide are encoded by two genes in tandem which share the same 3'-exon.

From a cDNA library prepared from venom glands of worker bees, clones encoding the precursors of apamin and MCD peptide have been isolated. The cDNAs are similar at the 5'-ends and identical in their 3'-regions. Analysis of the corresponding genes has revealed the existence of six exons separated by introns rich in A + T. Starting from the 5'-end, these exons are arranged in the following order: three exons of the mast cell-degranulating (MCD) peptide precursor, two exons of the gene for the apamin precursor, and finally a 3'-exon present in both cDNAs. This suggests that the bulk of the apamin gene resides in the third intron of the MCD peptide gene. Using inverse polymerase chain reaction, a segment of genomic DNA upstream of the first exon of the MCD precursor gene was obtained. The sequence of this segment shows 81% identity to the DNA sequence preceding the first exon of the apamin gene and both contain a putative TATA box. We thus propose that the mRNA encoding the apamin precursor originates from a primary transcript which starts in the third intron of the MCD peptide gene. Both cDNAs encode unusually small precursors comprising only 46 amino acids in case of apamin and 50 in the case of the MCD peptide.

Amino Acid Sequence↗

PubMed1996

Requirement of cysteine residues in exons 1-6 of the extracellular domain of the luteinizing hormone receptor for gonadotropin binding.

The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven-transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain.

Alternative Splicing↗

PubMed1997

The human CC chemokine receptor 5 (CCR5) gene. Multiple transcripts with 5'-end heterogeneity, dual promoter usage, and evidence for polymorphisms within the regulatory regions and noncoding exons.

Human CC chemokine receptor 5 (CCR5), mediates the activation of cells by the chemokines macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and RANTES, and serves as a fusion cofactor for macrophage-tropic strains of human immunodeficiency virus type 1. To understand the molecular mechanisms that regulate human CCR5 gene expression, we initiated studies to determine its genomic and mRNA organization. Previous studies have identified a single CCR5 mRNA isoform whose open reading frame is intronless. We now report the following novel findings. 1) Complex alternative splicing and multiple transcription start sites give rise to several distinct CCR5 transcripts that differ in their 5'-untranslated regions (UTR). 2) The gene is organized into four exons and two introns. Exons 2 and 3 are not interrupted by an intron. Exon 4 and portions of exon 3 are shared by all isoforms. Exon 4 contains the open reading frame, 11 nucleotides of the 5'-UTR and the complete 3'-UTR. 3) The transcripts appear to be initiated from two distinct promoters: an upstream promoter (PU), upstream of exon 1, and a downstream promoter (PD), that includes the "intronic" region between exons 1 and 3. 4) PU and PD lacked the canonical TATA or CAAT motifs, and are AT-rich. 5) PD demonstrated strong constitutive promoter activity, whereas PU was a weak promoter in all three leukocyte cell environments tested (THP-1, Jurkat, and K562). 6) We provide evidence for polymorphisms in the noncoding sequences, including the regulatory regions and 5'-UTRs. The structure of CCR5 was strikingly reminiscent of the overall structure of other chemokine/chemoattractant receptors, underscoring an important evolutionarily conserved function for a prototypical gene structure. This is the first description of functional promoters for any CC chemokine receptor gene, and we speculate that the complex pattern of splicing events and dual promoter usage may function as a versatile mechanism to create diversity and flexibility in the regulation of CCR5 expression.

Adult↗

PubMed2000

Identification of cis-elements regulating exon 15 splicing of the amyloid precursor protein pre-mRNA.

Alternative splicing of exon 15 of the amyloid precursor protein (APP) pre-mRNA generates two APP isoform groups APP(ex15) (containing exon 15) and L-APP (without exon 15), which show a cell-specific distribution in non-neuronal cells and neurons of rat. Both APP isoforms differ in regard to functional properties like post-translational modification, APP secretion, and proteolytic production of Abeta peptide from APP molecules. Since Abeta generation is an important factor in the development of Alzheimer's disease, one could anticipate that these major APP isoforms might contribute differentially to the mechanisms underlying neurodegeneration in Alzheimer's disease. In this study, we established an APP minigene system in a murine cell system to identify cis-acting elements controlling exon 15 recognition. A 12. 5-kilobase pair genomic fragment of the murine APP gene contained all cis-regulatory elements to reproduce the splicing pattern of the endogenous APP transcripts. By using this approach, two intronic cis-elements flanking exon 15 were identified that block the inclusion of exon 15 in APP transcripts of non-neuronal cells. Point mutation analysis of these intronic regions indicated that pyrimidine-rich sequences are involved in the splice repressor function. Finally, grafting experiments demonstrated that these regulatory regions cell-specifically enhance the blockage of a chimeric exon in the non-neuronal splicing system.

3T3 Cells↗

PubMed2000

Polypyrimidine track-binding protein binding downstream of caspase-2 alternative exon 9 represses its inclusion.

We have been using the caspase-2 pre-mRNA as a model system to study the importance of alternative splicing in the regulation of programmed cell death. Inclusion or skipping of a cassette-type exon in the 3' portion of this pre-mRNA leads to the production of isoforms with antagonistic activity in apoptosis. We previously identified a negative regulatory element (In100) located in the intron downstream of alternative exon 9. The upstream portion of this element harbors a decoy 3' acceptor site that engages in nonproductive commitment complex interactions with the 5' splice site of exon 9. This in turn confers a competitive advantage to the exon-skipping splicing pattern. Further characterization of the In100 element reveals a second, functionally distinct, domain located downstream from the decoy 3' acceptor site. This downstream domain harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the negative effect on exon 9 inclusion. Finally, we show that both domains of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3' splice site can modulate its activity. Our results thus reveal a complex composite element that regulates caspase-2 exon 9 alternative splicing through a novel mechanism.

Alternative Splicing↗

PubMed2001

A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene.

Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity.

3T3 Cells↗