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‣ A deficiência em piruvato-quinase (PK) na população portuguesa : estudos de genética molecular e populacional; Pyruvate-Kinase Deficiency in the Portuguese Population - A Molecular and Population Study

Manco, Licínio Manuel Mendes
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Tese de Doutorado
Português
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O estudo molecular dos doentes de naturalidade Portuguesa com anemia hemolítica por deficiência de piruvato-quinase (PK; EC 2.7.1.40) permitiu identificar 8 mutações diferentes no gene PKLR. Seis mutações são descritas pela primeira vez: 3 mutações missense [1435C>T(479Arg>Cys), 1670G>C(557Gly>Ala) e 1706G>A(569Arg>Gln)]; 2 mutações de splicing [IVS8(+2)T>G e IVS10(+1)G>C]; e a substituição –72A>G na região promotora. Foram ainda encontradas duas mutações missense [1456C>T(486Arg>Trp) e 993C>A(331Asp>Glu)], previamente descritas. As mutações missense resultam na troca de aminoácidos em zonas conservadas da subunidade PK-R, reduzindo a eficiência catalítica da enzima. A mutação 1435C>T ocorre no último dinucleótido do exão 10 e deverá afectar também o processo de splicing. A mutação -72A>G ocorre na sequência GATA mais próxima do exão 1, na região promotora R do gene PKLR, e é responsável por uma diminuição drástica da síntese de mRNA. A mutação IVS8(+2)T>G anula o local de splicing 5’ do intrão 8 e, devido à activação de um local alternativo de splicing, leva à inserção dos primeiros 20 nucleótidos do intrão 8 no mRNA. A mutação IVS10(+1)G>C anula o local de splicing 5’ no intrão 10...

‣ Organization of the human CD40L gene: implications for molecular defects in X chromosome-linked hyper-IgM syndrome and prenatal diagnosis.

Villa, A; Notarangelo, L D; Di Santo, J P; Macchi, P P; Strina, D; Frattini, A; Lucchini, F; Patrosso, C M; Giliani, S; Mantuano, E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/03/1994 Português
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Recently, CD40L has been identified as the gene responsible for X chromosome-linked hyper-IgM syndrome (HIGM1). CD40L on activated T cells from HIGM1 patients fails to bind B-cell CD40 molecules, and subsequent analysis of CD40L transcripts by reverse transcription PCR demonstrated coding region mutations in these patients. This approach, however, is of limited use for prenatal diagnosis of HIGM1 in the early-gestation fetus. In this report, we have defined the genomic structure of the CD40L gene, which is composed of five exons and four intervening introns. With this information, we have defined at the genomic level the CD40L gene abnormalities for three previously described HIGM1 patients who demonstrated clustered deletions in the CD40L coding region. These different deletions arose from three distinct mechanisms, including (i) a splice donor mutation with exon skipping, (ii) a splice acceptor mutation with utilization of a cryptic splice site, and (iii) a deletion/insertion event with the creation of a new splice acceptor site. In addition, we have performed prenatal evaluation of an 11-week-old fetus at risk for HIGM1. CD40L genomic clones provide a starting point for further studies of the genetic elements that control CD40L expression. Our knowledge of the CD40L gene structure will prove useful for the identification of additional mutations in HIGM1 and for performing genetic counseling about this disease.

‣ Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Chen, I T; Chasin, L A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1993 Português
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A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences...

‣ Analysis of Splice Variants of the Immediate-Early 1 Region of Human Cytomegalovirus

Awasthi, Sita; Isler, Jennifer A.; Alwine, James C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2004 Português
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The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces multiple mRNAs through differential splicing and polyadenylation. Reverse transcriptase PCR was used to characterize transcripts from exons 1, 2, 3, and 4 (immediate-early 1 [IE1]). The expected IE72 and IE19 mRNAs were detected, as well as two heretofore-uncharacterized transcripts designated IE17.5 and IE9. The IE72, IE19, and IE17.5 transcripts utilized the same 5′-splice site in exon 3. IE9 utilized a cryptic 5′-splice site within exon 3. The IE19, IE17.5, and IE9 transcripts all used different 3′-splice sites within exon 4. These spliced species occur in infected human foreskin fibroblast (HFF) cells, with accumulation kinetics similar to those of IE72 mRNA. IE19 and IE9 RNAs were much more abundant than IE17.5 RNA. Transfection of CV-1 cells with cDNAs resulted in IE19 and IE17.5 proteins detectable by antibodies to either N-terminal or C-terminal epitopes. No IE9 protein product has been detected. We have not been able to detect IE19, IE17.5, or IE9 proteins during infection of HFF, HEL, or U373MG cells. Failure to detect IE19 protein contrasts with a previous report (M. Shirakata, M. Terauchi, M. Ablikin, K. Imadome, K. Hirai, T. Aso, and Y. Yamanashi...

‣ Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford Progeria Syndrome

Scaffidi, Paola; Misteli, Tom
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A, one of the major architectural elements of the mammalian cell nucleus1–4. The HGPS mutation activates an aberrant cryptic splice site in the lamin A pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction of wild type lamin A levels3,4. Fibroblasts from HGPS patients have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from HGPS patients. Introduction of wild type lamin A protein does not rescue the cellular disease symptoms. The mutant lamin A mRNA and protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected, the dynamic properties of lamin A are restored, and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the application of small molecules to the correction of the premature aging phenotype in HGPS patients.

‣ Deletions within COL7A1 exons distant from consensus splice sites alter splicing and produce shortened polypeptides in dominant dystrophic epidermolysis bullosa.

Sakuntabhai, A; Hammami-Hauasli, N; Bodemer, C; Rochat, A; Prost, C; Barrandon, Y; de Prost, Y; Lathrop, M; Wojnarowska, F; Bruckner-Tuderman, L; Hovnanian, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1998 Português
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We describe two familial cases of dominant dystrophic epidermolysis bullosa (DDEB) that are heterozygous for deletions in COL7A1 that alter splicing, despite intact consensus splice-site sequences. One patient shows a 28-bp genomic deletion (6081del28) in exon 73 associated with the activation of a cryptic donor splice site within this exon; the combination of both defects restores the phase and replaces the last 11 Gly-X-Y repeats of exon 73 by a noncollagenous sequence, Glu-Ser-Leu. The second patient demonstrates a 27-bp deletion in exon 87 (6847del27), causing in-frame skipping of this exon; consensus splice sites, putative branch sites, and introns flanking exons 73 and 87 showed a normal sequence. Keratinocytes from the probands synthesized normal and shortened type VII collagen polypeptides and showed intracellular accumulation of type VII procollagen molecules. This first report of genomic deletions in COL7A1 in DDEB suggests a role for exonic sequences in the control of splicing of COL7A1 pre-mRNA and provides evidence that shortened type VII collagen polypeptides can alter, in a dominant manner, anchoring-fibril formation and can cause DDEB of differing severity.

‣ Aberrant 3′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

Vořechovský, Igor
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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The frequency distribution of mutation-induced aberrant 3′ splice sites (3′ss) in exons and introns is more complex than for 5′ splice sites, largely owing to sequence constraints upstream of intron/exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 3′ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 3′ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 3′ss was achieved by the maximum entropy model. Almost one half of aberrant 3′ss was activated by AG-creating mutations and ∼95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 3′ss was characterized by higher purine content than for authentic sites, particularly in position −3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position −11. A newly developed online database of aberrant 3′ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tools.

‣ Multifactorial Interplay Controls the Splicing Profile of Alu-Derived Exons▿ †

Ram, Oren; Schwartz, Schraga; Ast, Gil
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Exonization of Alu elements creates primate-specific genomic diversity. Here we combine bioinformatic and experimental methodologies to reconstruct the molecular changes leading to exon selection. Our analyses revealed an intricate network involved in Alu exonization. A typical Alu element contains multiple sites with the potential to serve as 5′ splice sites (5′ss). First, we demonstrated the role of 5′ss strength in controlling exonization events. Second, we found that a cryptic 5′ss enhances the selection of a more upstream site and demonstrate that this is mediated by binding of U1 snRNA to the cryptic splice site, challenging the traditional role attributed to U1 snRNA of binding the 5′ss only. Third, we used a simple algorithm to identify specific sequences that determine splice site selection within specific Alu exons. Finally, by inserting identical exons within different sequences, we demonstrated the importance of flanking genomic sequences in determining whether an Alu exon will undergo exonization. Overall, our results demonstrate the complex interplay between at least four interacting layers that affect Alu exonization. These results shed light on the mechanism through which Alu elements enrich the primate transcriptome and allow a better understanding of the exonization process in general.

‣ Noncanonical and canonical splice sites: a novel mutation at the rare noncanonical splice-donor cut site (IVS4+1A>G) of SEDL causes variable splicing isoforms in X-linked spondyloepiphyseal dysplasia tarda

Xiong, Feng; Gao, Jianjun; Li, Jun; Liu, Yun; Feng, Guoyin; Fang, Wenli; Chang, Hongfen; Xie, Jiang; Zheng, Haitao; Li, Tingyu; He, Lin
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Português
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X-linked spondyloepiphyseal dysplasia tarda can be caused by mutations in the SEDL gene. This study describes an interesting novel mutation (IVS4+1A>G) located exactly at the rare noncanonical AT–AC consensus splicing donor point of SEDL, which regained the canonical GT–AG consensus splicing junction in addition to several other rarer noncanonical splice patterns. The mutation activated several cryptic splice sites and generated the production of seven erroneous splicing isoforms, which we confirmed by sequencing of RT-PCR products and resequencing of cDNA clones. All the practical splice donors/acceptors were further assessed using FSPLICE 1.0 and SPL(M) Platforms to predict potential splice sites in genomic DNA. Subsequently, the expression levels of SEDL among the affected patients, carriers and controls were estimated using real-time quantitative PCR. Expression analyses showed that the expression levels of SEDL in both patients and carriers were decreased. Taken together, these results illustrated how disruption of the AT donor site in a rare AT–AC intron, leading to a canonical GT donor site, resulted in a multitude of aberrant transcripts, thus impairing exon definition. The unexpected splicing patterns resulting from the special mutation provide additional challenges and opportunities for understanding splicing mechanisms and specificity.

‣ Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function

Lemp, Nathan A.; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3′ splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3′ ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3′ ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

‣ Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases

Spinelli, Roberta; Pirola, Alessandra; Redaelli, Sara; Sharma, Nitesh; Raman, Hima; Valletta, Simona; Magistroni, Vera; Piazza, Rocco; Gambacorti-Passerini, Carlo
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Português
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Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC3 (NM_003786.3:c.1783-1G>A), KLHDC1 (NM_172193.1:c.568-2A>G), HOOK1 (NM_015888.4:c.1662-1G>A), SMAD9 (NM_001127217.2:c.1004-1C>T), and DNAH9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC3, HOOK1. In ABCC3 and HOOK1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK1. In GNAQ...

‣ Antisense Oligonucleotide Induction of Progerin in Human Myogenic Cells

Luo, Yue-Bei; Mitrpant, Chalermchai; Adams, Abbie M.; Johnsen, Russell D.; Fletcher, Sue; Mastaglia, Frank L.; Wilton, Steve D.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 03/06/2014 Português
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We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the ‘natural’ ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript...

‣ Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

Carothers, A M; Urlaub, G; Grunberger, D; Chasin, L A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1993 Português
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Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation...

‣ Structural requirements for selection of 5'- and 3' splice sites of group II introns.

Wallasch, C; Mörl, M; Niemer, I; Schmelzer, C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/06/1991 Português
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The group II intron bl1 in the gene for apocytochrome b in yeast mitochondrial DNA (COB) is self-splicing in vitro. It could recently be shown that self-splicing of this intron is fully reversible in vitro. In addition, intron integration is not restricted to parental exons, since the intron can also integrate into a foreign RNA. The position of insertion seems to be immediately 3' to a cryptic intron binding site 1 (IBS1). We confirmed and extended these results by sequencing 26 individual RNAs with transposed introns after reverse transcription and PCR amplification. Results show that intron integration into authentic exons is generally correct, but that integration into a foreign RNA is often inaccurate, i.e. insertion is one nt downstream or upstream of the 3' end of IBS1. This leads to the generation of 5' splice junctions of the new intron-harbouring 'preRNAs' with addition (or deletion) of a single A residue at the 3' end of IBS1. To investigate which structures help to define the position of 5'- and 3' cleavage, preRNAs of i) these clones with aberrant 5' splice junctions and ii) preRNAs with artificial hairpins between domains 5 and 6 of the intron were spliced under different reaction conditions. Results obtained let us conclude that i) branchpoint dependent 5' cleavage is directed by the 5' terminal G residue of the intron and...

‣ Congenital hypothyroidism due to mutations in the sodium/iodide symporter. Identification of a nonsense mutation producing a downstream cryptic 3' splice site.

Pohlenz, J; Rosenthal, I M; Weiss, R E; Jhiang, S M; Burant, C; Refetoff, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1998 Português
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A 12-yr-old hypothyroid girl was diagnosed at birth as athyreotic because her thyroid gland could not be visualized by isotope scanning. Goiter development due to incomplete thyrotropin suppression, a thyroidal radioiodide uptake of < 1%, and a low saliva to plasma ratio of 2.5 suggested iodide (I-) transport defect. mRNA isolated from her thyroid gland and injected into Xenopus oocytes failed to increase I- transport. Sequencing of the entire Na+/I- symporter (NIS) cDNA revealed a C to G transversion of nucleotide (nt) 1146 in exon 6, resulting in a Gln 267 (CAG) to Glu (GAG) substitution. This missense mutation produces an NIS with undetectable I- transport activity when expressed in COS-7 cells. Although only this missense mutation was identified in thyroid and lymphocyte cDNA, genotyping revealed that the proposita and her unaffected brother and father were heterozygous for this mutation. However, amplification of cDNA with a primer specific for the wild-type nt 1146 yielded a sequence lacking 67 nt. Genomic DNA showed a C to G transversion of nt 1940, producing a stop codon as well as a new downstream cryptic 3' splice acceptor site in exon 13, responsible for the 67 nt deletion, frameshift, and premature stop predicting an NIS lacking 129 carboxy-terminal amino acids. This mutation was inherited from the mother and present in the unaffected sister. Thus...

‣ Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.

Aroian, R V; Levy, A D; Koga, M; Ohshima, Y; Kramer, J M; Sternberg, P W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1993 Português
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The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.

‣ Splice-mediated insertion of an Alu sequence inactivates ornithine delta-aminotransferase: a role for Alu elements in human mutation.

Mitchell, G A; Labuda, D; Fontaine, G; Saudubray, J M; Bonnefont, J P; Lyonnet, S; Brody, L C; Steel, G; Obie, C; Valle, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1991 Português
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In studies of mutations causing deficiency of ornithine delta-aminotransferase (EC 2.6.1.13), we found an allele whose mature mRNA has a 142-nucleotide insertion at the junction of sequences from exons 3 and 4. The insert derives from an Alu element in ornithine delta-aminotransferase intron 3 oriented in the direction opposite to transcription (an "antisense Alu"). A guanine----cytosine transversion creates a donor splice site in this Alu, activating a cryptic acceptor splice site at its 5' end and causing splice-mediated insertion of an Alu fragment into the mature ornithine-delta-aminotransferase mRNA. We note that the complement of the Alu consensus sequence has at least two cryptic acceptor sites and several potential donor sequences and predict that similar mutations will be found in other genes.

‣ Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Português
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Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5...

‣ Possible role of splice acceptor site in expression of unspliced gag-containing message of Moloney murine leukemia virus.

Oshima, M; Odawara, T; Matano, T; Sakahira, H; Kuchino, Y; Iwamoto, A; Yoshikura, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1996 Português
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Moloney murine leukemia virus (MLV) having the gag coding region alone, G3.6, produced a low level of mRNA (1/10 of the wild-type level). Ligation of 441 nucleotides (nt) containing a splice acceptor (SA) site to the downstream portion of the remaining gag region restored the level of the unspliced message, simultaneously activating a cryptic splice donor (SD) site in the middle of the p30 coding region (between nt 1596 and 1597). Ligation of the 441 nt in the same site in the inverted orientation also increased the level of the unspliced message, activating the same SD site (between nt 1596 and 1597) and a new SA site just in front of the inserted 441 nt (between nt 4770 and 4771). Deletion or inversion of the 441-nt SA sequence from the wild-type MLV or from int in-frame deletion or int frameshift mutant MLVs of nearly full size resulted in the loss of spliced mRNA and concomitantly in a severe reduction of the unspliced mRNA, particularly at 37 degrees C. Deletion of the 5' SD site did not result in the reduction of the unspliced-mRNA level. When the gag region in G3.6 was replaced with a Neo(r) coding region, the level of expression was high. The data taken together suggest that the presence of an SA signal is necessary for high-level expression of unspliced mRNA encoding Gag or Gag-Pol.

‣ Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site

Aroian, Raffi V.; Levy, Adam D.; Koga, Makoto; Ohshima, Yasumi; Kramer, James M.; Sternberg, Paul W.
Fonte: Instituto de Tecnologia da Califórnia Publicador: Instituto de Tecnologia da Califórnia
Tipo: Article; PeerReviewed Formato: application/pdf
Publicado em /01/1993 Português
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The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of- function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.