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‣ Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

Romfo, Charles M.; Alvarez, Consuelo J.; van Heeckeren, Willem J.; Webb, Christopher J.; Wise, Jo Ann
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2000 Português
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Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5′ junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5′ junctions can compete effectively with the standard 5′ splice site if they are closer to the 3′ splicing signals. Taken together...

‣ Activation of cryptic 3′ splice sites within introns of cellular genes following gene entrapment

Osipovich, Anna B.; White-Grindley, Erica K.; Hicks, Geoffrey G.; Roshon, Michael J.; Shaffer, Christian; Moore, Jason H.; Ruley, H. Earl
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3′-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3′ splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3′ splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3′ splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3′ splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3′ splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3′ splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3′ processing and polyadenylation of cellular transcripts.

‣ Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

Jin, Y; Dietz, H C; Montgomery, R A; Bell, W R; McIntosh, I; Coller, B; Bray, P F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/10/1996 Português
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Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17.

‣ Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

Gantla, S; Bakker, C T; Deocharan, B; Thummala, N R; Zweiner, J; Sinaasappel, M; Roy Chowdhury, J; Bosma, P J; Roy Chowdhury, N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1998 Português
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Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites...

‣ Identification of a splice-site mutation in the human growth hormone-variant gene.

MacLeod, J N; Liebhaber, S A; MacGillivray, M H; Cooke, N E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1991 Português
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The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analysis of the elongated hGH-V mRNA demonstrated retention of the first 12 bases of intron 2, resulting from both a base substitution at the intron 2 splice-donor dinucleotide (GT----AT) and activation of a cryptic splice-donor site 12 bases downstream. Survey of a total of 60 additional chromosomes failed to reveal additional incidence of this mutation. The mutation, which we have designated hGH-Vintron 2, pos 1 (G----A), represents both an initial example of a nondeletional mutation within the hGH-V gene and corresponding structural alteration in the encoded hGH-V hormone.

‣ A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

Chillón, M; Dörk, T; Casals, T; Giménez, J; Fonknechten, N; Will, K; Ramos, D; Nunes, V; Estivill, X
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1995 Português
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mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients.

‣ Allele-specific recognition of the 3′ splice site of INS intron 1

Kralovicova, Jana; Vorechovsky, Igor
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Português
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Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined by INS variants that control the splicing efficiency of intron 1. The adenine allele at IVS1-6 (rs689), which rapidly expanded in modern humans, renders the 3′ splice site of this intron more dependent on the auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF). This interaction required both zinc fingers of the 35-kD U2AF subunit (U2AF35) and was associated with repression of a competing 3′ splice site in INS exon 2. Systematic mutagenesis of reporter constructs showed that intron 1 removal was facilitated by conserved guanosine-rich enhancers and identified additional splicing regulatory motifs in exon 2. Sequencing of intron 1 in primates revealed that relaxation of its 3′ splice site in Hominidae coevolved with the introduction of a short upstream open reading frame, providing a more efficient coupled splicing and translation control. Depletion of SR proteins 9G8 and transformer-2 by RNA interference was associated with exon 2 skipping whereas depletion of SRp20 with increased representation of transcripts containing a cryptic 3′ splice site in the last exon. Together...

‣ Use of a Cryptic Splice Site for the Expression of Huntingtin Interacting Protein 1 in Select Normal and Neoplastic Tissues

Graves, Chiron W.; Philips, Steven T.; Bradley, Sarah V.; Oravecz-Wilson, Katherine I.; Li, Lina; Gauvin, Alice; Ross, Theodora S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/2008 Português
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Huntingtin interacting protein 1 (HIP1) is a 116-kDa endocytic protein, which is necessary for the maintenance of several tissues in vivo as its deficiency leads to degenerative adult phenotypes. HIP1 deficiency also inhibits prostate tumor progression in mice. To better understand how deficiency of HIP1 leads to such phenotypes, we analyzed tumorigenic potential in mice homozygous for a Hip1 mutant allele, designated Hip1Δ3-5, which is predicted to result in a frame-shifted, nonsense mutation in the NH2 terminus of HIP1. In contrast to our previous studies using the Hip1 null allele, an inhibition of tumorigenesis was not observed as a result of the homozygosity of the nonsense Δ3-5 allele. To further examine the contrasting results from the prior Hip1 mutant mice, we cultured tumor cells from homozygous Δ3-5 allele–bearing mice and discovered the presence of a 110-kDa form of HIP1 in tumor cells. Upon sequencing of Hip1 DNA and message from these tumors, we determined that this 110-kDa form of HIP1 is the product of splicing of a cryptic U12-type AT-AC intron. This event results in the insertion of an AG dinucleotide between exons 2 and 6 and restoration of the reading frame. Remarkably, this mutant protein retains its capacity to bind lipids...

‣ Analysis of 30 Putative BRCA1 Splicing Mutations in Hereditary Breast and Ovarian Cancer Families Identifies Exonic Splice Site Mutations That Escape In Silico Prediction

Wappenschmidt, Barbara; Becker, Alexandra A.; Hauke, Jan; Weber, Ute; Engert, Stefanie; Köhler, Juliane; Kast, Karin; Arnold, Norbert; Rhiem, Kerstin; Hahnen, Eric; Meindl, Alfons; Schmutzler, Rita K.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 11/12/2012 Português
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Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed...

‣ A Novel Aberrant Splice Site Mutation in RAB23 Leads to an Eight Nucleotide Deletion in the mRNA and Is Responsible for Carpenter Syndrome in a Consanguineous Emirati Family

Ben-Salem, S.; Begum, M.A.; Ali, B.R.; Al-Gazali, L.
Fonte: S. Karger AG Publicador: S. Karger AG
Tipo: Artigo de Revista Científica
Português
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Carpenter syndrome is caused by mutations in the RAB23 gene that encodes a small GTPase of the Rab subfamily of proteins. Rab proteins are known to be involved in the regulation of cellular trafficking and signal transduction. Currently, only few mutations in RAB23 have been reported in patients with Carpenter syndrome. In this paper, we report the clinical features, molecular and functional analysis of 2 children from an Emirati consanguineous family with this syndrome. The affected children exhibit the typical features including craniosynostosis, typical facial appearance, polysyndactyly, and obesity. Molecular analysis of the RAB23 gene revealed a homozygous mutation affecting the first nucleotide of the acceptor splice site of exon 5 (c.482-1G>A). This mutation affects the authentic mRNA splicing and activates a cryptic acceptor site within exon 5. Thus, the erroneous splicing results in an eight nucleotide deletion, followed by a frameshift and premature termination codon at position 161 (p.V161fsX3). Due to the loss of the C-terminally prenylatable cysteine residue, the truncated protein will probably fail to associate with the target cellular membranes due to the absence of the necessary lipid modification. The p.V161fsX3 extends the spectrum of RAB23 mutations and points to the crucial role of prenylation in the pathogenesis of Carpenter syndrome within this family.

‣ BAP1 Missense Mutation c.2054 A>T (p.E685V) Completely Disrupts Normal Splicing through Creation of a Novel 5’ Splice Site in a Human Mesothelioma Cell Line

Morrison, Arianne; Chekaluk, Yvonne; Bacares, Ruben; Ladanyi, Marc; Zhang, Liying
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 01/04/2015 Português
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BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val), identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3’ end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1) a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5’ splice site (GU), which resulted in the deletion of 4 base pairs and presumably protein truncation; 2) a variety of alternative splicing products that led to retention of different introns: introns 14–16; introns 15–16; intron 14 and intron 16; 3) partial intron 14 and 15 retentions caused by activation of alternative 3’ splice acceptor sites (AG) in the introns. Taken together...

‣ Functional Characterization of the spf/ash Splicing Variation in OTC Deficiency of Mice and Man

Rivera-Barahona, Ana; Sánchez-Alcudia, Rocío; Viecelli, Hiu Man; Rüfenacht, Veronique; Pérez, Belén; Ugarte, Magdalena; Häberle, Johannes; Thöny, Beat; Desviat, Lourdes Ruiz
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 08/04/2015 Português
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The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5’ splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the “spf/ash” mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably...

‣ Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations

Bourbon, M.; Duarte, M.A.; Alves, A.C.; Medeiros, A.M.; Marques, L.; Soutar, A.K.
Fonte: BMJ Publishing Group Publicador: BMJ Publishing Group
Tipo: Artigo de Revista Científica
Publicado em /05/2009 Português
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Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A...

‣ Sensitivity of splice sites to antisense oligonucleotides in vivo.

Sierakowska, H; Sambade, M J; Schümperli, D; Kole, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1999 Português
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A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

‣ Accurate selection of a 5' splice site requires sequences within fibronectin alternative exon B.

Kuo, B A; Norton, P A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1999 Português
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Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5' splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5' splice sites in vitro. PRE-mediated 5' splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3' splice site can partially rescue proper exon B 5' splice site selection. In addition, we found that PRE 5' splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.

‣ Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae.

Chanfreau, G; Legrain, P; Dujon, B; Jacquier, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/06/1994 Português
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The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities. Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns. We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/. Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide. These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection.

‣ Human thiopurine methyltransferase pharmacogenetics. Kindred with a terminal exon splice junction mutation that results in loss of activity.

Otterness, D M; Szumlanski, C L; Wood, T C; Weinshilboum, R M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1998 Português
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Thiopurine methyltransferase (TPMT) catalyzes S-methylation of thiopurine drugs such as 6-mercaptopurine. Large variations in levels of TPMT activity in human tissue can result from a common genetic polymorphism with a series of alleles for low activity. This polymorphism is an important factor responsible for large individual variations in thiopurine toxicity and therapeutic efficacy. We now report a new variant allele, TPMT*4, that contains a G--> A transition that disrupts the intron/exon acceptor splice junction at the final 3' nucleotide of intron 9, the terminal intron of the TPMT gene. This new allele cosegregated within an extended kindred with reduced TPMT activity. We attempted to determine the mechanism(s) by which the presence of TPMT*4 might result in low enzyme activity. Although very few mature transcripts derived from allele TPMT*4 were detected, the mutation did lead to generation of at least two aberrant mRNA species. The first resulted from use of a novel splice site located one nucleotide 3' downstream from the original splice junction. That mRNA species contained a single nucleotide deletion and a frameshift within exon 10, the terminal exon of the gene. The second novel mRNA species resulted from activation of a cryptic splice site located within intron 9...

‣ In vitro splicing of adenovirus E1A transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3' splice site.

Gattoni, R; Schmitt, P; Stevenin, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/03/1988 Português
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During the analysis of the in vitro alternative splicing of the natural E1A transcript of adenovirus, other minor reactions were detected (Schmitt et al., 1987, Cell 50, 31-39). We report here their characterization. The first reaction concerns the excision of a 216 nucleotide intron delineated by the 9S 5' splice site and a 3' splice site 216 nucleotides downstream. It can occur on the premRNA transcript and the 13S and 12S mRNA species. Strikingly, the reaction uses one of 3 branch points located 51, 55 or 59 residues upstream of the 3' splice site, a distance which is unusually long since all the branch points mapped up to now are located between 18-37 nucleotides of the 3' splice site. The dramatic accumulation of the corresponding lariat intermediates, likely related to this long spacing indicates that the second splicing step is relatively unefficient. The second kind of reaction analysed is a cryptic splicing which uses a 3' splice site generated by the junction of the 13S mRNA exons, and leads to the formation of psi 12S and psi 9S mRNAs. In vitro, this reaction occurs only from a 13S mRNA transcript, and not from the 13S mRNA newly formed in the splicing assay, consistent with what has been observed in vivo. Thus, both the well known alternative and the minor reactions occurring in vivo from E1A premRNA and mRNAs are detected in vitro...

‣ Exon skipping by mutation of an authentic splice site of c-kit gene in W/W mouse.

Hayashi, S; Kunisada, T; Ogawa, M; Yamaguchi, K; Nishikawa, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/03/1991 Português
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The murine mutation dominant white spotting (W) is in the proto-oncogene, c-kit. The receptor tyrosine kinase encoded by this gene has pleiotropic effects on murine development including hemopoietic cells, pigment cells, and germ cells. In this study, mutation in W homozygous mouse was identified as a single base substitution (GT----AT) at the 5'-splice donor site of the exon which encodes the transmembrane domain. Two types of aberrant exon skipping resulted from this mutation, occurred in a tissue specific manner. Either transcript lost the exon coding for transmembrane region and therefore the product might not be functional for signal transduction. Any unusual cryptic splice sites were not activated by this mutation as beta-globin gene in beta-thalassaemia. In addition, twelve base pair sequence of the 3'-end of the exon prior to the exon coding for transmembrane domain was found to be alternatively spliced. These findings should provide the genetic base for not only the receptor function but the splicing mechanism.

‣ Yeast pre-mRNA splicing requires a minimum distance between the 5' splice site and the internal branch acceptor site.

Thompson-Jäger, S; Domdey, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1987 Português
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We have generated several deletions within the intron of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses. Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing. In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences. Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.