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Results for “Aberrant splicing”

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PubMed1990

The simian virus 40 small-t intron, present in many common expression vectors, leads to aberrant splicing.

Polymerase chain reaction analysis was used to identify aberrant splicing of the simian virus 40 small-t intron present in pRSVcat. We examined factors governing the selection and relative use of aberrant 5' splice sites derived from the chloramphenicol acetyltransferase-coding region. Our results indicated that transcripts from virtually any cDNA positioned upstream of the small-t intron could contain alternative 5' splice sites and therefore be subject to deletions within the protein-coding region.

Animals↗

PubMed1994

Construction of a novel database containing aberrant splicing mutations of mammalian genes.

To explore the rules for mammalian splice-site selection using a statistical approach, we constructed an aberrant splicing database containing an extensive collection of mammalian genetic disease mutations (90 genes, 209 mutations). From this database, we confirmed that: (1) more than 90% of mutations either destroy or create the splice-site consensus sequences; (2) the number of mutations mapped at individual residues in the splice-site regions roughly correlates to their conservation degrees in the consensus sequences; (3) about half of the observed aberrant splicing is exon skipping, while intron retention is rarely observed; (4) almost all of the major cryptic sites, activated by mutations, are mapped within an about 100-nt region from the authentic splice sites. Furthermore, we found that: (5) mutations are observed more frequently in the 5' splice-site region than in the 3' splice site region; (6) splice sites that are newly created by mutations are located upstream from the authentic splice sites. Hopefully, these observations will be used as rules for constructing a more effective prediction system of exon sequences.

Consensus Sequence↗

PubMed1998

Developmentally regulated muscle type-specific alternative splicing of the COOH-terminal variable region of fast skeletal muscle troponin T and an aberrant splicing pathway to encode a mutant COOH-terminus.

Distinct from the cardiac and slow skeletal muscle troponin Ts, an alternative RNA splicing-generated COOH-terminal variable region exists in the fast skeletal muscle troponin T. Mutually exclusive splicing of exon 16 and 17 encoded sequence into the mature mRNA produces the alpha- and beta-isoform, respectively. By cloning and sequence analysis of large numbers of fast troponin T cDNAs, we have quantitatively demonstrated that expression of the exon 16-encoded structure is mature fast muscle-specific (its utilization ranges from null in neonatal mouse muscles to 97% in adult chicken pectoralis), indicating a functional adaptation to the contractile feature of muscle. An aberrant splicing of this variable region to exclude both exons 16 and 17 from the mRNA was found in neonatal mouse skeletal muscle by cloning and sequencing characterization of a full length fTnT cDNA. The unusual splicing of exon 18 and exon 15 in the mRNA sequence results in not only a deletion of the exon 16/17 segment but also a shift of the downstream translation reading frame to produce a troponin T polypeptide with mutant COOH-terminus. Similar to an abnormal splicing of cardiac troponin T caused by cis-mutation and a dominant allele causing human familial hypertrophic cardiomyopathy, this trans-factor-determined aberrant mRNA splicing pathway generates a truncated troponin T molecule lacking the developmentally regulated fast muscle-specific COOH-terminal domain, indicating potential etiopathological significance.

Amino Acid Sequence↗

PubMed1997

Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours.

Intragenic deletions of TSG101, the human homolog of a mouse gene (tsg101) that acts to suppress malignant cell growth, were reported in human breast tumours. We screened TSG101 for somatic mutations in DNA and RNA samples isolated from a variety of common human malignancies, EBV-immortalised B-cells, and normal lung parenchyma. Intragenic TSG101 deletions in RNA transcripts were frequently found in all types of samples. Analysis of DNA failed to show genomic rearrangements corresponding to transcripts containing deletions in the same samples. The breakpoints of most transcript deletions coincide with genuine or cryptic splice site sequences, suggesting that they result from alternative or aberrant splicing. A similar spectrum of transcript deletions has previously been described in the putative tumour suppressor gene FHIT. We analysed FHIT in the same series of RNA samples and detected truncated FHIT transcripts frequently in both tumour and normal tissues. In addition, transcripts from TSG101, FHIT and seven other genes were analysed in RNA isolated from normal peripheral blood lymphocytes. Large TSG101 and FHIT intragenic transcript deletions were detected and these appeared to be the predominant transcript in 'aged' lymphocytes. Similar alterations were not detected in transcripts of the other genes which were analysed. Our findings demonstrate that truncated TSG101 and FHIT transcripts are commonly detected in both normal and malignant tissues and that a significant fraction of these are likely to be the result of aberrant splicing. While we cannot exclude that alterations in TSG101 and FHIT occur during cancer development, our data indicate that in this context the commonly observed transcript abnormalities are misleading.

Acid Anhydride Hydrolases↗

PubMed2006

Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing.

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model.

Numerous missense mutations in human BRCA1 gene have been linked to predisposition to breast cancer. However, the functional significance of the majority of these mutations remains unknown. We have examined the molecular basis for three such cancer-causing mutations. The first mutation, a T-->G transversion in codon 64, is predicted to change a conserved cysteine residue to glycine in the RING finger domain of the 1863 amino acid BRCA1 protein. Using a humanized mouse model we demonstrate that this missense mutation actually results in a functionally null protein. This striking result occurs because the single base alteration generates a new 5' splice site in exon 5 and also disrupts a putative exonic splicing enhancer motif. Consequently, the normal splice donor site is disrupted and an internal cryptic splice site is activated. This results in a 22-nucleotide deletion and the aberrant transcript is predicted to encode a severely truncated protein consisting of only 63 amino acids. To identify other missense mutations in BRCA1 that may result in aberrant splicing, we screened various mutations using the Genscan program. We demonstrate that at least two other missense mutations in codons 1495 and 1823 result in aberrant splicing due to the possible disruption of cis-acting splicing regulatory elements. In conclusion, our study demonstrates for the first time the application of a humanized mouse model for functional analysis of human mutations in mice and also shows the need for a careful examination of the functional consequences of single base alterations and single nucleotide polymorphisms identified in human disease-causing genes.

Alternative Splicing↗

PubMed2003

Glanzmann's thrombasthenia due to a point mutation within intron 10 results in aberrant splicing of the beta3 gene.

Glanzmann's thrombasthenia (GT) is an hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. We attempted to identify genetic defects responsible for a case of GT in Korea. The patient was a 6-year-old boy who had suffered from hemorrhage and purpura. The cDNAs of alphaIIb and beta3 were amplified by reverse transcription (RT)-PCR and were sequenced. RT-PCR of COS7 cells transfected with Exontrap vectors containing the beta3 genomic DNA fragments were performed to verify an aberrant splicing. Transient expression of alphaIIbbeta3 on transfected-COS7 cells was determined by flow cytometry, and the presence of alphaIIbbeta3 was confirmed by immunoprecipitation. We discovered an abnormality with the insertion of 38 bp between exon 10 and exon 11, including a stop codon, in beta3 cDNA. Sequence analysis of genomic DNA showed a point mutation, G-->T, at the position 29 107 of intron 10. RT-PCR analyses of COS7 cells transfected with Exontrap vector containing the mutant beta3 gene revealed that the point mutation at the position 29 107 of intron 10, G-->T, was responsible for an aberrant splicing in the beta3 gene. The transfection experiments and immunoprecipitation revealed the absence of alphaIIbbeta3 in the COS7 cells transfected with mutant gene. The mutation at the position 29 107 of intron 10, G-->T, in the beta3 genomic DNA was found to induce an aberrant splicing and to be responsible for GT in this patient.

Alternative Splicing↗

PubMed1997

Human CD46 aberrant splicing in transgenic mice.

RNA analysis of mice transgenic for human CD46 reveals almost undetectable levels of the expected transcript and the accumulation of a 900 nt shorter species. cDNA cloning and sequence analysis of this variant demonstrate an aberrant splicing of the transgene RNA. This aberrant splicing is present at low levels in cells that normally express the gene, suggesting that the transgene is lacking regulatory elements influencing the abundance of the shorter transcript.

3T3 Cells↗

PubMed2006

Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

Albinism, Ocular↗

PubMed1997

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.

Alternative Splicing↗

PubMed2000

Reversal of aberrant splicing of beta-thalassaemia allele (IVS-2-654 C-->T) by antisense RNA expression vector in cultured human erythroid cells.

The antisense fragment targeting the aberrant splice sites of the beta-thalassaemia allele, IVS-2-654 C-->T (beta654), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the beta654 mutant pretranscript in cultured beta654 erythroid cells by the lipofectin-mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the beta654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19 (day 0 after transfection) to 0.58 (day 8) in beta654/beta654 homozygous erythroid cells, and from 0.45 (day 0) to 0.83 (day 8) in beta654/betaA heterozygous erythroid cells, as determined by the ratio of normally spliced beta-globin transcript over total beta-globin transcript. Correspondingly, the ratios of globin chain biosynthesis (beta/alpha) increased from 0.16 (day 0) to 0.52 (day 8) in beta654/beta654 erythroid cells, and from 0.39 (day 0) to 0.84 (day 8) in beta654/betaA erythroid cells. Antisense RNA had no significant effect on the splicing pattern in betaA/betaA erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence). In addition, we did not observe side-effects on cytological features after the introduction of pCMVA. All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of beta654 mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.

Adult↗

PubMed2002

Cardiac troponin T variants produced by aberrant splicing of multiple exons in animals with high instances of dilated cardiomyopathy.

Adult cardiac muscle normally expresses a single cardiac troponin T (cTnT). As a potential pathogenic mechanism for turkey dilated cardiomyopathy, the splice-out of a normally constitutive exon generates an additional low molecular weight cTnT with altered conformation and function. We further found that aberrant splicing of cTnT also occurs in several mammals correlating to dilated cardiomyopathy. Skipping of the same exon as that in the turkey was found in the canine cTnT. Splice-out of the adjacent exon 6 occurred in the guinea pig cTnT. Retention of the embryonic exon 5 was found in the cTnT of cat, dog, and guinea pig. These aberrant splicing variants significantly altered the structure of cTnT to sustain functional effects as that in the myopathic turkey cTnT. The genomic sequence of canine cTnT gene shows no specific alterations. However, the alternative splicing patterns of canine cTnT are different in developing cardiac and skeletal muscles, suggesting abnormality of trans-regulatory factors. Transgenic expression of the aberrant cTnT variants resulted in contractile changes in mouse cardiomyocytes. The findings support the hypothesis that thin filament heterogeneity due to the co-expression of alternatively spliced cTnT variants may desynchronize myocardial contraction and contribute to the pathogenesis and pathophysiology of cardiomyopathy and heart failure.

Alternative Splicing↗

PubMed1999

Aberrant splicing of the Drosophila melanogaster phenylalanine hydroxylase pre-mRNA caused by the insertion of a B104/roo transposable element in the Henna locus.

We report the insertion of the transposable element B104 in the Phenylalanine hydroxylase gene of the Drosophila mutant Henna-recessive 3. Its presence alters the Phenylalanine hydroxylase splicing pattern, producing at least two aberrant mRNAs which contain part of the B104 sequence interrupting the coding region. This aberrant splicing is provoked by the use of a cryptic donor site encoded by the B104 3' long terminal repeat in combination with either the gene intron 3 acceptor site or a novel acceptor site generated by the target duplication caused by transposition. One of them, referred as mRNA type 1, encodes a truncated protein that could be predictably non-functional. In mRNA type 2, in spite of a 42 nt insertion, the Phenylalanine hydroxylase reading frame is not altered and it would encode for a protein with 14 extra amino acids which would be able to account for the low enzyme activity detected in this mutant. These results demonstrated that Henna locus encodes the enzyme phenylalanine hydroxylase providing direct evidence of its participation in pteridine synthesis. Moreover, it constitutes an example of the ability of transposable elements to generate protein variation in populations with the evolutionary consequences that this implies.

Alternative Splicing↗

PubMed2004

Two intronic mutations cause 17-hydroxylase deficiency by disrupting splice acceptor sites: direct demonstration of aberrant splicing and absent enzyme activity by expression of the entire CYP17 gene in HEK-293 cells.

To date, only two among 46 mutations in the CYP17 gene cause 17-hydroxylase deficiency (17OHD) by disrupting mRNA splice donor sites. We studied two subjects with intronic CYP17 mutations: a compound heterozygote for Y329D plus an AG to CG substitution at the 3' end of intron 2, and a homozygote for a TTTT deletion near the 3' end of intron 3. We hypothesized that both mutations caused 17OHD by disrupting splice acceptor sites. To prove this mechanism, the entire CYP17 genes (wild type and both mutations) were amplified, subcloned into pcDNA3, and expressed in HEK-293 cells. The mRNA derived from the wild-type CYP17 gene was correctly spliced and translated into active enzyme, as shown by the correct sequence in the RT-PCR products and by the 17-hydroxylation of progesterone. In contrast, cells expressing the mutant genes had no 17-hydroxylase activity. The mRNA derived from the AG to CG mutation used the first AG in exon 3 as the splice acceptor site, shifting the reading frame and introducing a stop codon. RNA derived from the TTTT deletion skipped exon 4 entirely, deleting 29 amino acids in-frame. Our data show that these are the first two 17OHD cases resulting from mutations that alter splice acceptor sites. These studies also demonstrate the feasibility of expressing the entire CYP17 gene, with simultaneous protein and RNA analysis, as a general methodology for characterizing how intronic CYP17 mutations cause 17OHD.

Adult↗

PubMed2004

Metals accelerate production of the aberrant splicing isoform of the presenilin-2.

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.

Alternative Splicing↗

PubMed1999

Stress-induced aberrant splicing of TSG101: association to high tumor grade and p53 status in breast cancers.

The TSG101 gene, identified through insertional mutagenesis, is localized in a region that exhibits LOH in human cancers, suggesting that TSG101 might be a tumor suppressor gene. Numerous studies have then shown the presence of abnormal transcripts in various tumors which appear to result from aberrant splicing of the gene, rather than from intragenic deletions. Moreover, many studies demonstrated that these aberrantly spliced transcripts were not found in matched normal tissues. We have analysed TSG101 transcripts in 85 breast cancer samples and found that abnormal splicing of the gene is tightly correlated with tumor grade and p53 mutation. In addition, stress induced the appearance of these abnormal transcripts in primary lymphocytes. Hence, TSG101 splicing defects, while unrelated to the oncogenic process per se, could reflect the cellular environment of the tumor cells. The proposed role of stress and hypoxia to select p53 mutant cells could account for the tight association with p53 status.

Alternative Splicing↗

PubMed2004

Delineation of the mechanisms of aberrant splicing caused by two unusual intronic mutations in the RSK2 gene involved in Coffin-Lowry syndrome.

Coffin-Lowry syndrome (CLS) is caused by mutations in the RSK2 gene encoding a protein kinase of the Ras signalling pathway. We have studied two point mutations which cause aberrant splicing but do not concern the invariant GT or AG nucleotides of splice sites. The first, an A-->G transition at position +3 of the 5' splice site of exon 6, results in vivo and in vitro in exon skipping and premature translation termination. The natural 5' splice site, although intrinsically weak, is not transactivated under normal conditions. Consequently, replacement of an A/U by a G/U base pairing with U1 snRNA reduces its strength below a critical threshold. The second mutation, an A-->G transition 11 nt upstream of exon 5, creates a new AG near the natural 3' splice site. In vitro this synthetic 3' AG is used exclusively by the splicing machinery. In vivo this splicing event is also observed, but is underestimated because the resulting RSK2 mRNA contains premature stop codons which trigger the nonsense-mediated decay process. We show that a particular mechanism is involved in the aberrant splicing of exon 5, implying involvement of the natural 3' AG during the first catalytic step and the new 3' AG during the second step. Thus, our results explain how these mutations cause severe forms of CLS.

Base Sequence↗

PubMed1999

Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides.

The CFTR splicing mutation 3849 + 10 kb C --> T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 + 10 kb C --> T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2'-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose- and sequence-dependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders.

3T3 Cells↗