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‣ Redefinition of exon 7 in the COL1A1 gene of type I collagen by an intron 8 splice-donor-site mutation in a form of osteogenesis imperfecta: influence of intron splice order on outcome of splice-site mutation.

Schwarze, U; Starman, B J; Byers, P H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1999 Português
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Most splice-site mutations lead to a limited array of products, including exon skipping, use of cryptic splice-acceptor or -donor sites, and intron inclusion. At the intron 8 splice-donor site of the COL1A1 gene, we identified a G+1-->A transition that resulted in the production of several splice products from the mutant allele. These included one in which the upstream exon 7 was extended by 96 nt, others in which either intron 8 or introns 7 and 8 were retained, one in which exon 8 was skipped, and one that used a cryptic donor site in exon 8. To determine the mechanism by which exon-7 redefinition might occur, we examined the order of intron removal in the region of the mutation by using intron/exon primer pairs to amplify regions of the precursor nuclear mRNA between exon 5 and exon 10. Removal of introns 5, 6, and 9 was rapid. Removal of intron 8 usually preceded removal of intron 7 in the normal gene, although, in a small proportion of copies, the order was reversed. The proportion of abnormal products suggested that exon 7 redefinition, intron 7 plus intron 8 inclusion, and exon 8 skipping all represented products of the impaired rapid pathway, whereas the intron-8 inclusion product resulted from use of the slow intron 7-first pathway. The very low-abundance cryptic exon 8 donor site product could have arisen from either pathway. These results suggest that there is commitment of the pre-mRNA to the two pathways...

‣ Oriented Scanning Is the Leading Mechanism Underlying 5′ Splice Site Selection in Mammals

Borensztajn, Keren; Sobrier, Marie-Laure; Duquesnoy, Philippe; Fischer, Anne-Marie; Tapon-Bretaudière, Jacqueline; Amselem, Serge
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional...

‣ Identification of alternative 5′/3′ splice sites based on the mechanism of splice site competition

Xia, Huiyu; Bi, Jianning; Li, Yanda
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
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Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5′/3′ splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified ∼70% of the splice sites into alternative and constitutive, as well as ∼80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.

‣ Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition

Královičová, Jana; Vořechovský, Igor
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together...

‣ A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome

Gong, Qiuming; Zhang, Li; Moss, Arthur J.; Vincent, G. Michael; Ackerman, Michael J.; Robinson, Jeffrey C.; Jones, Melanie A.; Tester, David J.; Zhou, Zhengfeng
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the mechanism of the hERG splice site mutation 2398+1G>C and the genotype-phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of index patients and using minigenes expressed in HEK293 cells and neonatal rat ventricular myocytes. RT-PCR analysis revealed that the 2398+1G>C mutation disrupted the normal splicing and activated a cryptic splice donor site in intron 9, leading to the inclusion of 54 nt of the intron 9 sequence in hERG mRNA. The cryptic splicing resulted in an in-frame insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. In patch clamp experiments the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that the mutant expressed an immature form of hERG protein that failed to reach the plasma membrane. Coexpression of the mutant and wild-type channels led to a dominant negative suppression of wild-type channel function by intracellular retention of heteromeric channels. Our results demonstrate that 2398+1G>C activates a cryptic site and generates a full-length hERG protein with an insertion of 18 amino acids...

‣ New Splice Site Acceptor Mutation in AIRE Gene in Autoimmune Polyendocrine Syndrome Type 1

Mora, Mireia; Hanzu, Felicia A.; Pradas-Juni, Marta; Aranda, Gloria B.; Halperin, Irene; Puig-Domingo, Manuel; Aguiló, Sira; Fernández-Rebollo, Eduardo
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 02/07/2014 Português
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Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset...

‣ Activation of cryptic splice sites in murine sarcoma virus-124 mutants.

de Mars, M; Cizdziel, P E; Murphy, E C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1990 Português
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We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVts110 retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVts110 or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site...

‣ Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information.

Hebsgaard, S M; Korning, P G; Tolstrup, N; Engelbrecht, J; Rouzé, P; Brunak, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/09/1996 Português
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Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local prediction of splice sites, is refined by rules based on splice site confidence values, prediction scores, coding context and distances between potential splice sites. In this approach, the prediction of splice sites mutually affect each other in a non-local manner. The combined approach drastically reduces the large amount of false positive splice sites normally haunting splice site prediction. An analysis of the errors made by the networks in the first step of the method revealed a previously unknown feature, a frequent T-tract prolongation containing cryptic acceptor sites in the 5' end of exons. The method presented here has been compared with three other approaches, GeneFinder, Gene-Mark and Grail. Overall the method presented here is an order of magnitude better. We show that the new method is able to find a donor site in the coding sequence for the jelly fish Green Fluorescent Protein, exactly at the position that was experimentally observed in A.thaliana transformants. Predictions for alternatively spliced genes are also presented...

‣ Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing.

Chang, J S; McPheeters, D S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/2000 Português
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Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue. In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.

‣ Identification of a specific exon sequence that is a major determinant in the selection between a natural and a cryptic 5' splice site.

Domenjoud, L; Gallinaro, H; Kister, L; Meyer, S; Jacob, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1991 Português
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The first intron of the early region 3 from adenovirus type 2 contains a cryptic 5' splice site, Dcr1, 74 nucleotides downstream from the natural site D1. The cryptic site can be activated when the natural site is inactivated by mutagenesis. To investigate the basis for selection between a natural and a cryptic 5' splice site, we searched for cis-acting elements responsible for the exclusive selection of the natural site. We show that both the relative intrinsic strength of the sites and the sequence context affect the selection. A 120-nucleotide segment located at the 3' end of exon 1 enhances splicing at the proximal site D1; in its absence the two sites are used according to their strength. Thus, three cis-acting elements are involved in the silencing of the cryptic site: the sequence of D1, the sequence of Dcr1, and an upstream exonic sequence. We show that the exonic element folds, in solution, into a 113-nucleotide-long stem-loop structure. We propose that this potential stem-loop structure which is located 6 nucleotides upstream of the exon 1-intron junction is responsible for the preferential use of the natural 5' splice site.

‣ An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

Lou, H; Yang, Y; Cote, G J; Berget, S M; Gagel, R F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1995 Português
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Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.

‣ Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.

Suh, E R; Waring, R B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1990 Português
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It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

‣ Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals.

Wiebauer, K; Herrero, J J; Filipowicz, W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1988 Português
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The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts...

‣ Exon definition may facilitate splice site selection in RNAs with multiple exons.

Robberson, B L; Cote, G J; Berget, S M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1990 Português
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Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons...

‣ The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.

Barfod, E T; Cech, T R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1989 Português
Relevância na Pesquisa
48.97101%
Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore...

‣ Interactions across exons can influence splice site recognition in plant nuclei.

McCullough, A J; Baynton, C E; Schuler, M A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1996 Português
Relevância na Pesquisa
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In vivo analyses of cis-acting sequence requirements for pre-mRNA splicing in tobacco nuclei have previously demonstrated that the 5' splice sites are selected by their position relative to AU-rich elements within plant introns and by their degree of complementarity to the U1 small nuclear RNA. To determine whether the presence of adjacent introns affects 5' splice site recognition in plant nuclei, we have analyzed the in vivo splicing patterns of two-intron constructs containing 5' splice site mutations in the second intron. These experiments indicated that the splice site selection patterns in plant nuclei are defined primarily by sequences within the intron (intron definition) and secondarily by weak interactions across exons (exon definition). The effects of these secondary interactions became evident only when mutations in the downstream 5' splice site decreased its functionality and differed depending on the availability of cryptic splice sites close to the mutant site. In beta-conglycinin chimeric transcripts containing multiple cryptic 5' splice sites, the presence of an intact upstream intron significantly increased splicing at the downstream 5' splice sites in a polar fashion without activating exon skipping. In a natural beta-conglycinin transcript...

‣ Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

Bradley, K; Cavaco, B; Bowl, M; Harding, B; Young, A; Thakker, R
Fonte: BMJ Group Publicador: BMJ Group
Tipo: Artigo de Revista Científica
Publicado em /08/2005 Português
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More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g→a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant.

‣ 3' splice site selection in dicot plant nuclei is position dependent.

Lou, H; McCullough, A J; Schuler, M A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1993 Português
Relevância na Pesquisa
49.07901%
In contrast to mammalian and yeast systems, the mechanism for intron recognition and splice site selection in plant pre-mRNAs is poorly understood. Splice site sequences and putative branchpoint sequences are loosely conserved in plant introns compared with other eukaryotes. Perhaps to compensate for these variations, plant introns are significantly richer in adenosine and uridine residues than are their adjacent exons. To define elements critical for 3' splice site selection in dicotyledonous plant nuclei, pre-mRNA transcripts containing intron 3 of the maize Adh1 gene were expressed in Nicotiana benthamiana nuclei by using an autonomously replicating plant expression vector. Using a series of intron rearrangements which reposition the 3' intron-exon border, we demonstrate that the normal 3' splice site is defined in a position-dependent manner and that cryptic 3' splice sites within the intron are masked by the presence of a functional downstream 3' splice site. Disruption of the AU-rich elements upstream from the normal 3' splice site indicates that multiple AU elements between -66 and -6 cooperatively define the 3' boundary of the intron. These results are consistent with a model for plant intron recognition in which AU-rich elements spread throughout the length of the intron roughly define the intron boundaries by generating strong AU transition points. Functional 3' splice sites located downstream from these AU-rich sequences are preferentially selected over sites embedded within them.

‣ AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster.

McCullough, A J; Schuler, M A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1993 Português
Relevância na Pesquisa
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cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco...

‣ Role of the branch site/3'-splice site region in adenovirus-2 E1A pre-mRNA alternative splicing: evidence for 5'- and 3'-splice site co-operation.

Ulfendahl, P J; Kreivi, J P; Akusjärvi, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/02/1989 Português
Relevância na Pesquisa
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The adenovirus E1A gene encodes five overlapping mRNAs which are processed by alternative RNA splicing from a common pre-mRNA. To characterize cis-acting sequence elements which are of importance for the alternative 5'-splice site selection deletion and substitution mutants within the intron that is common to all E1A mRNAs were constructed. Deletion of the wild-type E1A branch site/polypyrimidine tract resulted in activation of a functionally redundant sequence located within an A/T rich sequence just upstream of the normal E1A lariat branch site. Removal of both regulatory sequences abolished in vivo splicing completely and did not lead to activation of cryptic 3'-splice sites at other locations in the E1A pre-mRNA. Furthermore we show that the sequence around the E1A branch site/3'-splice site region may have a more direct effect on the efficiency by which the alternative E1A 5'-splice sites are selected. Replacing the E1A branch site/3'-splice site region with the corresponding sequence from the second intron of the rabbit beta-globin gene or the first intron of the major late transcription unit resulted in drastic changes in E1A 5'-splice site selection. For example, with the E1A/beta-globin hybrid gene the 9S mRNA became the most abundant E1A mRNA to accumulate. This contrasts with the wild-type E1A gene in which almost undetectable levels of 9S mRNA were produced in transient expression assays. Our results strongly suggest that a cooperative interaction between 5'- and 3'-splice sites on a pre-mRNA determines the outcome of alternative splicing.