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At least 343 records · Page 19Linked to original sources

PubMed2001

Genomic organization of the human thyroglobulin gene: the complete intron-exon structure.

OBJECTIVE: In order to complete the knowledge of the genomic organization of the human thyroglobulin gene, the present work was designed to establish the intron-exon organization from exon 24 to exon 35 and to construct a more complete physical map of the gene. DESIGN: Screening of two genomic libraries, and subsequent restriction mapping, hybridization and sequencing were used to characterize the recombinant phages. METHODS: Two human genomic DNA libraries were screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. The Long PCR method was used to amplify the genomic DNA region containing exon 24. Intron-exon junction sequences were determined by using the Taq polymerase-based chain termination method. RESULTS: We isolated and characterized five lambda phage clones that include nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25-35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequenced from the amplified fragment. In total, 8010 intronic bases were analyzed. CONCLUSIONS: The present study shows that the five phages isolated and the amplified fragment include 59.4 kb genomic DNA, covering 1446 nucleotides of exonic sequence distributed over 12 exons, from exon 24 to exon 35. Using previous studies and our current data, 220 kb of the human thyroglobulin gene was analyzed, a physical map was constructed, and all exon-intron junctions were sequenced and correlated with the different domains of the protein. In summary, the thyroglobulin gene contains 48 exons ranging in size from 63 nucleotides to 1101 nucleotides.

Base Sequence↗

PubMed2001

[Study on polymorphism of D gene exons among RhD-negative Chinese Han population].

OBJECTIVE: To explore the genomic structure of 8 exons in D gene of RhD(-) Chinese Han population. METHODS: Polymerase chain reaction-sequence specific primers(PCR-SSP) were used to study genomic DNA from 50 samples of RhD(-) Chinese Han donors. Exons 2, 3, 4, 5, 6, 7, 9, 10 of RHD gene and exons 1, 2, 4, 5 of RHCE gene were specifically amplified, also intron 4 of them was amplified. RESULTS: Phenotypes of the 50 RhD(-) donors were: 22 ccdee, 22 Ccdee, 3 CcdEe, 3 CcdEe. The 8 exons of RHD gene were completely absent in 25 donors with ccdee or ccdEe phenotype, while polymorphisms of D gene exons were found in 25 donors with Ccdee or CcdEe phenotype: the presence of all 8 exons of D gene in 9 donors, the absence of the 8 exons in 7 donors, the presence of exon 2 in 5 donors, the presence of exon 6 in 3 donors and the presence of exons 2, 6, 10 in 1 donor were demonstrated. CONCLUSION: Polymorphisms of RHD gene exons were present among RhD(-) Chinese Han blood donors. The 8 exons of RHD gene were completely absent in donors with Rhesus cc phenotype, while 5 polymorphisms of RHD gene exons were found in donors with Rhesus Cc phenotype. The discrepancy of the RHD gene in RhD(-) individuals between Chinese Hans and Caucasians indicates that care should be exercised by clinicians in the use of the RhD genotyping results.

China↗

PubMed1992

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.

Animals↗

PubMed1992

Human alpha 2(VI) collagen gene. Heterogeneity at the 5'-untranslated region generated by an alternate exon.

Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.

Amino Acid Sequence↗

PubMed1995

Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity.

A functional analysis of exon replacement mutations was performed in parallel with RNA-protein binding assays to gain insight into the role of the exon in alternative and simple splicing events. These results show that constitutive exons from unrelated genes contain strong signals that promote splicing in multiple sequence contexts by enhancing 3' splice site activity. A clue to the nature of the relationship between the exon and adjacent 3' splice site is indicated by the binding properties of exon variant RNAs when tested with different biochemical preparations of the essential splicing protein, U2AF. In the context of a complete nuclear extract, U2AF binding to the 3' splice site is stimulated by the presence of an adjacent constitutive exon. In contrast, highly purified HeLa U2AF binds equivalently to the exon variants under conditions in which differential polypyrimidine tract binding is evident. These results provide support for an assisted binding model in which positive-acting signals within exons, exon enhancers, direct the binding of accessory factors, which in turn increase the intrinsic affinity of U2AF for the adjacent 3' splice site. Further support for an assisted binding model is indicated by biochemical complementation of U2AF binding and by the localization of a novel exon enhancer, which, when introduced into a weak exon, stimulates splicing activity in parallel with U2AF binding. Immunoprecipitation analysis identifies the splicing factor, SC35, as a constituent of the exon enhancer binding complex. These results are discussed in the context of current models for functional exon-bridging interactions.

Base Sequence↗

PubMed1995

Isolation and characterization of the murine C-FGR genomic locus: exons IB-XII.

The c-fgr proto-oncogene is a member of the src family of intracellular protein tyrosine kinases. C-fgr is selectively expressed in hematopoietic cells, particularly in monocytes, neutrophils and natural killer cells. Although c-fgr is presumed to play a role in signal transduction, its normal function has not been completely elucidated. In contrast to all other members of the src family, the presumed murine and human c-fgr homologues have a low degree of homology in their amino terminal domains which likely mediate the association of c-fgr proteins with signalling complexes and other targets; murine and human c-fgr proteins exhibit other functional differences as well. In contrast to human C-FGR, the murine C-FGR genomic locus has not been characterized. We have now isolated and mapped three overlapping phage clones spanning 33 kb of the murine C-FGR genomic locus. Contained within these clones are 18 kb of DNA containing the C-FGR coding exons (exons II-XII), one 5' untranslated region exon (exon Ib) located 1.7 kb upstream of exon II, 6.5 kb of DNA upstream of exon Ib and 6.7 kb of DNA downstream of the polyadenylation site in exon XII. From exons Ib-XII, the exon-intron organization, exon-intron junctions and intron lengths of the murine and human C-FGR genomic loci are quite similar; with the exception of exon II, the exon lengths are also quite similar. Although these studies suggest that the murine and human C-FGR genes are in fact homologues, the organization of these genes upstream of exon Ib is quite divergent.

Animals↗

PubMed1997

The association of nonsense mutation with exon-skipping in hprt mRNA of Chinese hamster ovary cells results from an artifact of RT-PCR.

RT-PCR of RNA from CHO cells with nonsense mutations in the hprt gene frequently detects minor hprt mRNA species lacking one or more exons. Many nonsense mutants also contain greatly reduced concentrations of the major, normally spliced hprt mRNA. In this study, we examined the hypothesis that exon-deleted mRNAs are normal constituents of CHO cells, but are not detected in wild-type parental cells and most missense mutants because their amplification is suppressed by relatively high concentrations of normally spliced hprt mRNA. A protocol designed to specifically detect exon-deleted mRNAs was conducted using RNA from parental cells and identified all the exon-deleted species typical of nonsense mutants. Quantitative analysis of parental cell RNA measured these exon-deleted mRNAs at < or = 0.7% of the abundance of the full-sized species. Nonsense and missense mutants had comparable amounts of exon-deleted mRNAs, which varied both above and below parental concentrations. The relative concentrations of particular exon-deleted species could be explained by the location of nonsense mutations remaining in the mRNA or by structural effects of mutations on splicing. Exon-deleted mRNAs were detected by RT-PCR when the concentration of the most abundant exon-deleted species was > or = 2% of the full-length mRNA. This occurred for mutants with nonsense mutations in internal exons. RT-PCR conditions were shown to suppress the amplification of exon-deleted species 40-fold when full-length mRNA was abundant, which occurred for parental lines and missense mutants. Our results verify that RT-PCR conditions can produce an artifactual association between nonsense mutation and exon-skipping when minor, exon-deleted mRNA is relatively enriched.

Animals↗

PubMed1997

Structural analysis of the mouse prosaposin (SGP-1) gene reveals the presence of an exon that is alternatively spliced in transcribed mRNAs.

SGP-1/prosaposin can be secreted or targeted to the lysosomes where it is processed into smaller saposins A, B, C, and D required for the hydrolysis of glycosphingolipids. The deficiency of saposins B and C results in variant forms of metachromatic leukodystrophy and Gaucher's disease, respectively, which are characterized by lysosomal storage of undegraded glycosphingolipids. A required step to correct these genetic defects, or to understand the targeting mechanism of SGP-1 to the lysosomes, or to the extracellular space as well as its interaction with specific glycosphingolipids, is the analysis of the gene encoding this protein. Thus our investigation dealt with the molecular cloning of the mouse SGP-1 gene. Sequence analysis revealed that the mouse SGP-1 gene consists of 15 exons ranging from nine base pairs to 298 base pairs and 14 introns, which ranged from 89 base pairs to >8 kb in length. Our data show that saposin A is encoded by the exons 3, 4, and 5, saposin B by exons 6, 7, 8, and 9, saposin C by exons 10 and 11, and saposin D by exons 12, 13, and 14. The translation start codon is located within exon 1, and the translation stop codon is located within exon 15. The exon/intron boundaries were in accordance to the AG/GT consensus sequences. Our data also revealed that the SGP-1 gene has an exon consisting of the nine base pairs (CAG GAT CAG) encoding the three amino acids of saposin B, which may be alternatively spliced in the SGP-1 mRNA. The presence of the different forms of alternatively spliced mRNAs in various tissues was analyzed by RT-PCR. This approach demonstrated that prosaposin mRNAs of brain, heart, and muscle contain the nine base pairs of exon 8, whereas the transcripts from testis, lung, pancreas, spleen, and kidney do not contain this exon 8. Sequence comparison between the human and mouse prosaposin showed that exon 11 of mouse SGP-1 consists of 279 base pairs, whereas the human prosaposin gene consists of 187 base pairs. The extra 93 base pairs encode 31 amino acids corresponding to a proline-rich region located between saposin C and saposin D in the mouse prosaposin molecule. Finally, the availability of these genomic clones provides a starting point for further studies on the genetic role of specific sequences on the structure and function of SGP-1/prosaposin and its derived saposin proteins. In conclusion, we cloned and sequenced the mouse prosaposin (SGP-1) gene. The structural analysis of this gene revealed the presence of an exon that is alternatively spliced in transcribed mRNAs in a tissue-specific manner.

Alternative Splicing↗

PubMed1995

Human choline acetyltransferase gene: localization of alternative first exons.

Two overlapping cosmids containing the 5' end of human choline acetyltransferase (ChAT) gene have been cloned. Using heterologous probes, we localized two alternative first exons homologous to rodent ChAT exons R and M (Misawa et al.: J Biol Chem 267:20392-20399, 1992). The sequence of rodent exon N was not conserved in the human gene. Northern blot analysis of mRNA purified from the human neuroepithelioma cell lines LA-N2 and MC-I-XC revealed that both exons R and M were transcribed in mRNA species of 6.0 and 2.5 kb. Only the 6-kb species was detected with both R- and M-specific probes in the neuroepithelioma cell line CHP126. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that the major mRNA species in MC-I-XC and CHP126 cells contained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT-PCR also allowed the characterization of a mRNA species containing exon R spliced to exon 1, but no species containing both exon R and the distal part of exon M could be detected. RT-PCR was also used to evidence an alternative exon (tentatively numbered exon 8) in the coding sequence.

Alternative Splicing↗

PubMed2005

A novel cryptic exon identified in the 3' region of intron 2 of the human dystrophin gene.

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), is the largest known human gene and is characterized by the huge size of its introns. Intron 2, the second largest intron, is 170-kb long and has been shown to include a 140-bp cryptic exon (exon 2a) in its 5' region. The rest of this intron has no known function. In this study, we find that another cryptic exon, located in the 3' region of intron 2, is activated in a promoter- or tissue-specific manner. An unknown 98-bp insertion precisely between exons 2 and 3 was identified in one of the dystrophin mRNAs from lymphocytes of a DMD patient with a duplication of exon 2. This 98-bp sequence, located in the 3' region of intron 2, was found to possess a branch point, acceptor and donor splice-site consensus sequences, and an exonic splicing enhancer sequence, and thus is a novel exon, which we named "exon 2b." In lymphocytes, exon 2b incorporation was detected in the muscle-specific, promoter-driven transcript. Five of 20 normal human tissue mRNAs, including cardiac and skeletal muscle mRNAs, were confirmed to contain a fragment extending from exon 1 to exon 2b by reverse transcription PCR amplification, indicating that exon 2b is activated in a tissue-specific manner. This provides a clue to a novel cause of dystrophinopathy.

3' Untranslated Regions↗

PubMed2004

An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy.

SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.

Amino Acid Motifs↗

PubMed2000

Identification of a novel human BCL-X promoter and exon.

BCL-XL is a key anti-apoptotic BCL-2 family protein that is widely expressed in human cancer cells and is induced in response to diverse survival signals. The translation initiation codon for BCL-XL is located in BCL-X exon II and previous analyses have indicated that BCL-XL RNAs initiate close to the start of exon II or additionally contain a non-coding first exon (exon IA) spliced to exon II. Using 5' RACE we have now identified a novel BCL-X non-coding exon (exon IB) which is spliced directly to exon II in place of exon IA. Exon IB-containing RNAs encoded BCL-XL and were detected in non-malignant lymphocytes and lymphoma cells from lymph node biopsies and were expressed at significant levels in cell lines derived from ovarian, colon and breast cancers. We identified two TATA-box sequences upstream of exon IB and demonstrated that surrounding genomic sequences contained strong promoter activity in lymphoma cells (approximately 300-fold active relative to controls). We have therefore identified a powerful new BCL-X promoter and a novel exon that contributes to BCL-XL expression.

Base Sequence↗

PubMed2001

Characterization of the human and mouse genes for the alpha subunit of type II prolyl 4-hydroxylase. Identification of a previously unknown alternatively spliced exon and its expression in various tissues.

Prolyl 4-hydroxylase (4-PH) catalyzes the formation of 4-hydroxyproline in -X-Pro-Gly- sequences and has a central role in the synthesis of all collagens. We report here on the cloning and characterization of the genes encoding the catalytic alpha(II) subunits of the human and mouse type II 4-PH [alpha(II)]2beta2 tetramers. The human and mouse genes are approximately 34.6 kb and 30.3 kb in size, respectively, and both consist of 16 exons. The translation initiation codons are located in exon 2, and the sizes of the exons consisting entirely of coding sequences are conserved in the two genes, varying from 54 to 240 bp, whereas the exons 1, containing the transcription initiation sites and 5' untranslated sequences, are 546 bp and 293 bp in the human and mouse, respectively. The sizes of the introns vary from 48 to 49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but they and introns 1 contain several motifs that may act as transcription factor binding sites, including those for Sox9, which regulates chondrocyte-specific expression of collagens II, IX and XI. Unlike the human alpha(I) gene, the alpha(II) genes do not contain an alternatively spliced exon homologous to exon 9. However, a novel mutually exclusively spliced alternative exon 12a was identified in both genes. The nucleotide and amino-acid sequence identities between the 60-bp exon 12a and 66-bp exon 12b are about 35% and 45%, respectively, in both human and mouse genes. PCR analyses showed that both types of exon 12 are expressed in all tissues studied, except for adult leukocytes that expressed only mRNAs containing exon 12b sequences. Insect cell expression studies showed that a recombinant alpha(II) subunit containing amino acids coded by exon 12a associated with the beta subunit to form a fully active enzyme tetramer.

Alternative Splicing↗

PubMedDate not supplied

Identification of various exon combinations of the ews/fli1 translocation: an optimized RT-PCR method for paraffin embedded tissue -- a report by the CWS-study group.

BACKGROUND: Chromosomal translocations t(11;22) (q24;q12) are characteristic of about 80-90 % of Ewing's sarcoma family of tumors [bone and soft tissue Ewing's sarcoma and peripheral neuroectodermal tumors (PNET)]. They generate ews/fli1 rearrangements showing great diversity in breakpoint exon combination. In about 5 % of Ewing's tumors, ews is fused to the erg gene at 21q22. The various chimeric proteins encoded may function as aberrant oncogenic transcription factors. These specific translocations can be used for exact molecular diagnosis in these poorly differentiated small round-cell tumors. Moreover, the prognostic relevance of different translocational variants has been previously suggested. Furthermore, the sensitive molecular detection of minimal metastatic and residual disease and its clinical significance can be evaluated. To address these questions more definitively in the large number of patients registered in multicenter studies, it is often necessary to access archival paraffin-embedded tumor tissue if no fresh or frozen tumor material is available for analysis by RT (reverse transcription)-PCR. Specific problems arise from formalin-fixed and paraffin-embedded tissue due to the degradation of RNA and insufficient extraction efficiency. Therefore, primer distance and product size are limited for successful PCR amplification. This conflicts with the requirement for identification of various possible exon combinations by PCR simultaneously using one single primer pair with larger distance. PATIENTS: We examined paraffin embedded soft part tumor tissue samples from 47 Ewing's tumor patients. Patients were treated according to either CWS (Cooperative Weichteilsarkomstudie, CWS-91 or CWS-96) or Euro-E.W.I.N.G. 99 therapy protocols. METHOD: We established a novel RT-PCR method, using 3 different exon specific sets of PCR primer pairs, selected according to the coding ews and fli1 nucleotide sequences (NCBI database), suitable for RT-PCR identification of variant ews/fli1 fusion transcripts in RNA isolated from formalin-fixed, paraffin-embedded tissue. For use in combination with ews -primer, an erg specific primer was selected to alternatively test for ews/erg fusion transcripts. As positive control for the integrity of isolated mRNA, we used the ubiquitously expressed gapdh transcript for RT-PCR amplification in each sample. RESULTS: In 31 cases (= 66 %) of 47 paraffin samples of Ewing's tumors analysed, gapdh control indicated adequate quality of RNA. In 16 cases no gapdh control fragment was amplifiable, nevertheless in 2 of these 16 samples distinct ews fusion products could be detected. In 23 cases we identified ews fusion transcripts. Thereof in 65 % ews exon 7 being fused to fli1 exon 6 (fusion type I), in 22 % to fli1 exon 5 (fusion type II). In 4 % each ews exon 10 being juxtaposed to fli1 either exon 6 or exon 5, respectively. An ews/erg fusion was detected in 4 % ( ews exon 7 fused to erg exon 6). In 10 samples, a gapdh fragment was amplified, but no ews/fli1 or - erg fusion transcript could be identified. The reference pathological review (I. L., Kiel, Germany) disproved the primary histopathology in 5 cases. CONCLUSIONS: Using our different sets of exon specific primer pairs, it was possible to detect 4 different breakpoints of ews/fli1 fusion transcripts and the ews/erg fusion by RT-PCR in RNA isolates from formalin-fixed, paraffin-embedded Ewing's tumor tissue. This method can be a very useful alternative in clinical situations (to ensure diagnosis and perform minimal metastatic and residual disease investigations) and in order to assess prognostic significance of translocation subtypes when no fresh tumor tissue is available.

Electrophoresis, Agar Gel↗

PubMed2004

Polypyrimidine tract-binding protein is involved in vivo in repression of a composite internal/3' -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA.

The Xenopus alpha(fast)-tropomyosin gene contains, at its 3' -end, a composite internal/3' -terminal exon (exon 9A9'), which is subjected to three different patterns of splicing according to the cell type. Exon 9A9' is included as a terminal exon in the myotome and as an internal exon in adult striated muscles, whereas it is skipped in nonmuscle cells. We have developed an in vivo model based on transient expression of minigenes encompassing the regulated exon 9A9' in Xenopus oocytes and embryos. We first show that the different alpha-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and constitute valuable tools to seek regulatory sequences involved in exon 9A9' usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9' in nonmuscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9'. We show using UV cross-linking and immunoprecipitation experiments that Xenopus PTB (XPTB) interacts with these PTB binding sites. Finally, we show that depletion of endogenous XPTB in Xenopus embryos using a morpholinobased translational inhibition strategy resulted in exon 9A9' inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9' and suggest that PTB could be a major actor in the repression of regulated 3' -terminal exon.

Actins↗

PubMed2005

Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb.

The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.

Alternative Splicing↗

PubMed1990

Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development.

During development of the rat central nervous system, neural cell adhesion molecule (NCAM) mRNAs containing in the extracellular domain a 30-bp alternative exon, here named VASE, replace RNAs that lack this exon. The presence of this alternative exon between previously described exons 7 and 8 changes the predicted loop structure of the derived polypeptide from one resembling an immunoglobulin constant region domain to one resembling an immunoglobulin variable domain. This change could have significant effects on NCAM polypeptide function and cell-cell interaction. In this report we test multiple rat tissues for the presence of additional alternative exons at this position and also examine the regulation of splicing of the previously described exon. To sensitively examine alternative splicing, polymerase chain reactions (PCRs) with primers flanking the exon 7/exon 8 alternative splicing site were performed. Four categories of RNA samples were tested for new exons: whole brain from embryonic day 11 to adult, specific brain regions dissected from adult brain, clonal lines of neural cells in vitro, and muscle cells and tissues cultured in vitro and obtained by dissection. Within the limits of the PCR methodology, no evidence for any alternative exon other than the previously identified VASE was obtained. The regulation of expression of this exon was found to be complex and tissue specific. Expression of the 30-bp exon in the heart and nervous system was found to be regulated independently; a significant proportion of embryonic day 15 heart NCAM mRNAs contain VASE while only a very small amount of day 15 nervous system mRNAs contain VASE. Some adult central nervous system regions, notably the olfactory bulb and the peripheral nervous system structures adrenal gland and dorsal root ganglia, express NCAM which contains very little VASE. VASE is undetectable in NCAM PCR products from the olfactory epithelium. Other nervous system regions express significant quantities of NCAM both with and without VASE. Clonal cell lines in culture generally expressed very little VASE. These results indicate that a single alternative exon, VASE, is found in NCAM immunoglobulin-like loop 4 and that distinct tissues and nervous system regions regulate expression of VASE independently both during development and in adult animals.

Amino Acid Sequence↗

PubMed2000

An alternative-exon database and its statistical analysis.

We compiled a comprehensive database of alternative exons from the literature and analyzed them statistically. Most alternative exons are cassette exons and are expressed in more than two tissues. Of all exons whose expression was reported to be specific for a certain tissue, the majority were expressed in the brain. Whereas the length of constitutive exons follows a normal distribution, the distribution of alternative exons is skewed toward smaller ones. Furthermore, alternative-exon splice sites deviate more from the consensus: their 3' splice sites are characterized by a higher purine content in the polypyrimidine stretch, and their 5' splice sites deviate from the consensus sequence mostly at the +4 and +5 positions. Furthermore, for exons expressed in a single tissue, adenosine is more frequently used at the -3 position of the 3' splice site. In addition to the known AC-rich and purine-rich exonic sequence elements, sequence comparison using a Gibbs algorithm identified several motifs in exons surrounded by weak splice sites and in tissue-specific exons. Together, these data indicate a combinatorial effect of weak splice sites, atypical nucleotide usage at certain positions, and functional enhancers as an important contribution to alternative-exon regulation.

Algorithms↗