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At least 127 records · Page 7Linked to original sources

PubMed2005

Exon 5 of the RET proto-oncogene: a newly detected risk exon for familial medullary thyroid carcinoma, a novel germ-line mutation Gly321Arg.

Familial medullary thyroid carcinoma (FMTC) is an autosomal dominant inherited disease, characterized by germ-line mutations in the RET proto-oncogene, mainly in exons 10 and 11, but also in exons 13, 14 and 15. Recently, mutations in exons 8 and 16 associated with FMTC were also described. In the herein presented study, single strand conformation polymorphism (SSCP) method for rapid screening of mutations in the RET proto-oncogene and fluorescent sequencing method were used. In one Czech family with FMTC, we have identified a novel missense point mutation of the RET proto-oncogene in exon 5, that results in substitution of arginine by glycine at codon 321 in the cadherin-like domain of ret protein. It seems that this mutation causes FMTC as no other mutation was found in the classical risk exons (10, 11, 13, 14, 15 and 16) of the RET proto-oncogene. The mutation cosegregates with medullary thyroid carcinoma (MTC) or C cell hyperplasia (CCH) in two patients; two other family members are mutation carriers without clinical signs of MTC so far. To improve the diagnosis of FMTC, analysis of exon 5 of the RET proto-oncogene should be considered in families with no identified classical RET mutations.

Adult↗

PubMed2002

A novel cryptic exon in intron 3 of the dystrophin gene was incorporated into dystrophin mRNA with a single nucleotide deletion in exon 5.

The dystrophin gene, which is mutated in Duchenne muscular dystrophy, is the largest human gene. A full spectrum of the gene transcripts has not been fully elucidated yet, although two cryptic exons have so far been identified in the 5' region of the dystrophin gene. Here, a novel dystrophin mRNA containing a 62-nucleotide insertion between exons 3 and 4 was identified in lymphocytes from a Japanese Duchenne muscular dystrophy patient with a single nucleotide deletion in exon 5. The inserted 62-nucleotide sequence was found to be homologous to part of intron 3 and it was revealed that the insertion possessed branch point and both acceptor and donor splice site consensus sequences perfectly. Therefore, the 62-bp insertion sequence was considered to be a novel exon and was designated as exon 3a. However, this insertion was not present in the patient's muscle and 12 different normal tissues that were screened. The physiological role of the novel cryptic exon remains to be clarified.

Alternative Splicing↗

PubMed2005

Skipping of exon 2 and exons 2 plus 3 of HMG-CoA lyase (HL) gene produces the loss of beta sheets 1 and 2 in the recently proposed (beta-alpha)8 TIM barrel model of HL.

HMG-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine catabolism. We report a new Spanish patient who bears the frequent nonsense mutation G109T (Mediterranean mutation). This mutation can produce aberrant splicing with three mRNA variants: one of the expected size, the second with deletion of exon 2, and the third with deletion of exons 2 and 3. Recently our group proposed a 3D model for human HL containing a (beta-alpha)(8) (TIM) barrel structure. We have studied the effect of the deletions of exon 2 and exons 2 plus 3 on the proposed HL model. Exon 2 skipping led to the loss of beta-sheet 1, and the skipping of exons 2 and 3 caused the disappearance of alpha helix 1 and beta-sheets 1 and 2.-

Base Sequence↗

PubMed2004

Protein domains correlate strongly with exons in multiple eukaryotic genomes--evidence of exon shuffling?

We conducted a multi-genome analysis correlating protein domain organization with the exon-intron structure of genes in nine eukaryotic genomes. We observed a significant correlation between the borders of exons and domains on a genomic scale for both invertebrates and vertebrates. In addition, we found that the more complex organisms displayed consistently stronger exon-domain correlation, with substantially more significant correlations detected in vertebrates compared with invertebrates. Our observations concur with the principles of exon shuffling theory, including the prediction of predominantly symmetric phase of introns flanking the borders of correlating exons. These results suggest that extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.

Amino Acid Sequence↗

PubMed2001

A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H.

An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3'-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 approximately 60 nucleotides downstream of 3'-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5' extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3'-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two U-to-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3'-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF(35) for binding to the exon sequence flanking 3'-splice site A3. This binding results in the inhibition of splicing at 3'-splice site A3.

Base Sequence↗

PubMed2003

Mutations in tau gene exon 10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2 beta.

Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2 beta protein. The interaction between Tra2 beta and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2 beta protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2 beta, suggesting that Tra2 beta may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies.

Alternative Splicing↗

PubMed2005

Negative transcriptional modulation and silencing of the bi-exonic Rnf35 gene in the preimplantation embryo. Binding of the CCAAT-displacement protein/Cux to the untranslated exon 1 sequence.

Previous works have indicated promiscuous transcription from the zygotic genome immediately after fertilization. The mouse Rnf35 gene is bi-exonic in structure and is transcribed in the preimplantation embryo until it is permanently silenced at the blastocyst stage of development. We have previously shown that Rnf35 transcription is positively regulated by the nuclear factor Y. Using the uniquely permissive Chinese hamster ovary-K1 cell line in transient transfection assays, we demonstrate in this work that the Rnf35 promoter was negatively modulated by a cis-cognate repressor element, designated as the downstream exon 1 repressor, or DER, residing between +72 and +95 in the untranslated exon 1 of the Rnf35 gene. Simultaneous mutagenesis of the two half-sections, DER1 and DER2, of the DER sequence was required for derepression suggesting participation of multiple proteins in the DER-dependent transcriptional repression. Electrophoretic mobility shift assays demonstrated that the 3'-half of DER (DER2) was targeted by the repressor CCAAT-displacement protein (CDP)/Cux. Chromatin immunoprecipitation experiments further demonstrated in vivo CDP-DER association in the blastocyst and the 8.5 day embryo. Furthermore, the DER-dependent repression was partially relieved in vivo in co-transfection with an antisense CDP construct. Transcription of the Cdp gene was shown to first occur between the eight-cell and the blastocyst stages, correlating and possibly explaining the onset of Rnf35 silencing at the blastocyst stage. Taken together, our results suggest that the evolutionarily acquired exon 1 of Rnf35, and possibly exon 1 of other similarly structured bi-exonic early embryonic genes, contributes to transcriptional modulation and silencing in the developing mouse embryo.

Animals↗

PubMed2005

Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites.

As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional dystrophin proteins and potential conversion of severe DMD into milder Becker muscular dystrophy (BMD) phenotypes. We have previously described 37 exon-internal AONs that induce skipping of 14 DMD exons in human control myotube cultures. Here, we report 77 new AONs, effectively targeting an additional 21 exons. Of the 114 AONs thus far tested, 72 (67%) were effective. AON design initially was based on a partial overlap with predicted open secondary structures in the target RNA. We have analyzed various AON and target exon parameters in retrospect. Interestingly, we observed significantly higher SF2/ASF, SC35, and SRp40 values (as predicted by ESEfinder) for effective AONs when compared with ineffective AONs. In addition, the distance to the 3' splice site was significantly smaller for effective AONs. No other significant correlations were observed. Our results suggest that effective exon-internal AONs primarily act by blocking SR binding sites (which often correspond to open structures) and that ESEfinder may be used to refine AON design for DMD and other genes.

Base Sequence↗

PubMed2002

Nucleocytoplasmic transfer of the NF2 tumor suppressor protein merlin is regulated by exon 2 and a CRM1-dependent nuclear export signal in exon 15.

The neurofibromatosis 2 protein merlin is a classical tumor suppressor protein. Germline mutations predispose to the development of schwannomas, meningiomas and ependymomas. Merlin has been implicated in cellular migration and adhesion. This function is reflected in its subcellular localization at the plasma membrane and known interacting partners. Merlin has been regarded as an exception in not exerting a functional role within the nucleus as other tumor suppressors do. Here, we show that detection of wild-type protein in the nucleus is a rare event. However, splicing out of exon 2 leads to unrestricted entry into the nucleus. Skipping of adjacent exon 3 has no comparable effect ruling out an unspecific effect due to misfolding of the 4.1/JEF domain. Exon 2 functions as a cytoplasmic retention factor as it is able to confer sole cytoplasmic localization to a GFP fusion protein. Nuclear entry of merlin is thus regulated by alternative splicing within the 4.1/JEF domain and analogous to band 4.1 protein. Merlin's ability to enter the nucleus is complemented by a full nuclear-cytoplasmic shuttle protein with a functional Rev-type nuclear export sequence (NES) within exon 15 that facilitates export via the CRM1/exportin pathway. Deletion of this NES or treatment with the CRM1-specific inhibitor leptomycin B leads to overall nuclear accumulation of merlin isoforms missing exon 2. A cellular function different to the wild-type protein is implied for naturally occurring splice variants lacking exon 2. A putative effect of merlin as a transcriptional regulator and identification of nuclear binding partners remains to be elucidated.

3T3 Cells↗

PubMed1990

Beta-globin transcripts carrying a single intron with three adjacent nucleotides of 5' exon are efficiently spliced in vitro irrespective of intron position or surrounding exon sequences.

To examine the role of exon sequences and intron position in the splicing of an mRNA precursor, we prepared series of sense or anti-sense transcripts of human beta-globin cDNA in which a cassette containing the beta-globin first intron was inserted into one of seven unusual positions. The intron cassette consisted of the intron alone (ml), the intron with three adjacent base pairs of the 5' exon (MI), or the intron with both 5' and 3' exon sequences. All these transcripts were examined in an in vitro splicing system with a HeLa cell nuclear extract. The sense transcripts carrying MI cassette were spliced efficiently and independently of the intron position, except when the 3' exon was too short. The anti-sense transcripts carrying MI cassette produced significantly less spliced products than did those of the sense transcripts. This was mostly because of the instability of the anti-sense transcripts, and the actual splicing efficiency was similar to that seen in the sense transcripts. Sense or anti-sense transcripts carrying ml cassette were spliced to various extents depending on the surrounding sequences. The results indicate that only three nucleotides of the 5' exon are required as specific exon sequences in the splicing of an mRNA precursor carrying a single intron, and that the intron position does not significantly affect the splicing efficiency in vitro.

Base Sequence↗

PubMed1992

Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons.

Circularly permuted group I intron precursor RNAs, containing end-to-end fused exons which interrupt half-intron sequences, were generated and tested for self-splicing activity. An autocatalytic RNA can form when the primary order of essential intron sequence elements, splice sites, and exons are permuted in this manner. Covalent attachment of guanosine to the 5' half-intron product, and accurate exon ligation indicated that the mechanism and specificity of splicing were not altered. However, because the exons were fused and the order of the splice sites reversed, splicing released the fused-exon as a circle. With this arrangement of splice sites, circular exon production was a prediction of the group I splicing mechanism. Circular RNAs have properties that would make them attractive for certain studies of RNA structure and function. Reversal of splice site sequences in a context that allows splicing, such as those generated by circularly permuted group I introns, could be used to generate short defined sequences of circular RNA in vitro and perhaps in vivo.

Anabaena↗

PubMed1993

The exon trapping assay partly discriminates against alternatively spliced exons.

A cosmid containing eight exons of the gene coding for the microtubule-associated tau protein was subjected to the exon trapping assay. All the constitutive exons contained in the cosmid (4, 5, 7 and 9) were efficiently captured regardless of size. Of the four alternatively spliced exons, three (3, 4A and 8) were not isolated by the assay, but the behavior of exon 6 depended on the identity of its flanking exons.

Alternative Splicing↗

PubMed1997

Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2.

We have previously demonstrated that an exon splicing silencer (ESS) is present within human immunodeficiency virus type 1 (HIV-1)tat exon 2. This 20 nucleotide (nt) RNA element acts selectively to inhibit splicing at the upstream 3'splice site (3'ss #3) flanking this exon. In this report, we have used in vitro splicing of mutated RNA substrates to determine the sequences necessary and sufficient for the activity of the ESS. The activity of the ESS within tat exon 2 maps to a 10 nt core sequence CUAGACUAGA. This core sequence was sufficient to inhibit splicing when inserted downstream from the 3'ss of the heterologous Rous sarcoma virus src gene. Mutagenesis of the interspersed purines in the polypyrimidine tract of the tat exon 2 3'ss to pyrimidines resulted in a significant increase in splicing efficiency indicating that 3'ss#3 is suboptimal. The ESS acts to inhibit splicing at the optimized 3'splice sites of both the HIV-1 tat and RSV src constructs but with a reduced efficiency compared to its effect on suboptimal 3'splice sites. The results indicate that both the ESS and a suboptimal 3'splice site act together to control splicing at the 3'splice site flanking at exon 2.

Avian Sarcoma Viruses↗

PubMed2001

Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Nonsense-mediated messenger RNA (mRNA) decay, or NMD, is a critical process of selective degradation of mRNAs that contain premature stop codons. NMD depends on both pre-mRNA splicing and translation, and it requires recognition of the position of stop codons relative to exon-exon junctions. A key factor in NMD is hUpf3, a mostly nuclear protein that shuttles between the nucleus and cytoplasm and interacts specifically with spliced mRNAs. We found that hUpf3 interacts with Y14, a component of post-splicing mRNA-protein (mRNP) complexes, and that hUpf3 is enriched in Y14-containing mRNP complexes. The mRNA export factors Aly/REF and TAP are also associated with nuclear hUpf3, indicating that hUpf3 is in mRNP complexes that are poised for nuclear export. Like Y14 and Aly/REF, hUpf3 binds to spliced mRNAs specifically ( approximately 20 nucleotides) upstream of exon-exon junctions. The splicing-dependent binding of hUpf3 to mRNAs before export, as part of the complex that assembles near exon-exon junctions, allows it to serve as a link between splicing and NMD in the cytoplasm.

3' Untranslated Regions↗

PubMed1997

U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon.

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, we have taken advantage of our previous observation that U1 small nuclear RNAs (snRNAs) which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, we show that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter was duplicated suggest an optimal distance of greater than 72 nucleotides. We have also found that inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS. Our results are discussed in the context of models to explain constitutive splicing of small exons in nature.

Base Sequence↗

PubMed2004

Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis.

AIMS: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. METHODS: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. RESULTS: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains. CONCLUSIONS: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.

Brazil↗

PubMed2004

A novel type 2A von Willebrand factor mutation located at the last nucleotide of exon 26 (3538G>A) causes skipping of 2 nonadjacent exons.

In this manuscript, we describe a case of type 2A von Willebrand disease (VWD) caused by the novel heterozygous G>A transition at nucleotide 3538, which should result in the putative, nonconservative substitution of G1180R. This mutation was reproduced by site-directed mutagenesis; however, the recombinant mutant protein was efficiently secreted from cells and assembled correctly into multimers. Because the substitution is located at the last nucleotide of exon 26, the patient's platelet von Willebrand factor (VWF) mRNA was analyzed and 3 transcripts were observed: the normal transcript without the 3538G>A transition, a transcript with the in-frame deletion of exon 26, and a transcript with the in-frame deletions of exons 23 and 26. These deletion VWF cDNA constructs were created and the resulting recombinant proteins were analyzed following transfection into COS-7 cells. Cotransfection results demonstrate that the exon-skipped transcripts led to intracellular retention, and the levels of VWF antigen (VWF:Ag) produced by these constructs were as follows: del23/26 A transition at nucleotide (nt) 3538 does not result in the expression of the G1180R missense mutation, but rather leads to exon skipping, which is the pathogenic basis of the patient's phenotype. This is the first report of a coding region mutation resulting in the skipping of 2 nonadjacent exons.

Adolescent↗

PubMed1995

Identification of alternative exons, including a novel exon, in the tyrosine kinase receptor gene Etk2/tyro3 that explain differences in 5' cDNA sequences.

Protein tyrosine kinase transmembrane receptors trigger signal transduction cascades upon ligand binding, resulting in cellular proliferation, differentiation, differentiation inhibition or apoptosis depending upon the cell target. The ETK2/TYRO3 receptor is a tyrosine kinase expressed in embryonic stem cells, brain and testis that has recently been cloned by several groups. Analysis of cDNA clones isolated from several tissues shows 2 isoforms of the Etk2/tyro3 gene product that result from usage of alternative exons near the 5' end of the gene. In addition, our data suggest that a third alternative exon is positioned between these two alternative exons. This novel exon encodes yet another isoform that predicts a unique amino-terminal protein sequence. The alternative exons (exons 2A, 2B and 2C), predict three isoforms with different initiation codons, signal sequences and lengths. The existence of these multiple isoforms may be important for protein processing, translocation, or function.

Alternative Splicing↗