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Results for “Exon skipping”

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PubMed2001

Mild muscular dystrophy due to a nonsense mutation in the LAMA2 gene resulting in exon skipping.

Nonsense mutations outside the splicing consensus sequence have been reported to cause skipping of the nonsense-containing exon in several human diseases. We describe, for the first time, nonsense-mediated exon skipping in the laminin alpha2 (LAMA2) gene. Two siblings from a consanguineous family had altered expression of the laminin alpha2 chain and moderate clinical manifestations. In both we identified the new nonsense mutation Arg744Stop, which we expected to result in a totally non-functional polypeptide. However, analysis of the transcript revealed skipping of exon 15, containing the mutation, even though the consensus sequences for splicing at both ends of the exon and the beginning of intron 15 were unaltered. Exon skipping restored the open reading frame of the mutant transcript and resulted in a truncated protein. In cases where the genetic findings do not elucidate the phenotype, mRNA analysis is necessary to clarify the primary effect of mutations. Our findings also point to the necessity of immunochemical screening for expression of laminin alpha2 chain in atypical dystrophic adults as well as children.

Adult↗
PubMed2025

U2AF1 mutations rescue deleterious exon skipping induced by KRAS mutations.

The mechanisms by which somatic mutations of splicing factors, such as U2AF1S34F in lung adenocarcinoma, contribute to cancer pathogenesis are not well understood. Here, we used prime editing to modify the endogenous U2AF1 gene in lung adenocarcinoma cells and assessed the resulting impact on alternative splicing. These analyses identified KRAS as a key target modulated by U2AF1S34F. One specific KRAS mutation, G12S, generates a cryptic U2AF1 binding site that leads to skipping of KRAS exon 2 and generation of a non-functional KRAS transcript. Expression of the U2AF1S34F mutant reverts this exon skipping and restores KRAS function. Analysis of cancer genomes reveals that U2AF1S34F mutations are enriched in KRASG12S-mutant lung adenocarcinomas. A comprehensive analysis of splicing factor/oncogene mutation co-occurrence in cancer genomes also revealed significant co-enrichment of KRASQ61R and U2AF1I24T mutations. Experimentally, KRASQ61R mutation leads to KRAS exon 3 skipping, which in turn can be rescued by the expression of U2AF1I24T. Our findings provide evidence that splicing factor mutations can rescue splicing defects caused by oncogenic mutations. More broadly, they demonstrate a dynamic process of cascading selection where mutational events are positively selected in cancer genomes as a consequence of earlier mutations.

Journal Article↗
PubMed2000

Exon skipping mutation in the COL9A2 gene in a family with multiple epiphyseal dysplasia.

Previous linkage analysis (Briggs, M.D., Choi, H.-C., Warman, M.L. et al., 1994. Genetic mapping of a locus for multiple epiphyseal dysplasia (EDM 2) to a region of chromosome 1 containing a type IX collagen gene. Am. J. Hum. Genet. 55, 678-684) in a large English family with multiple epiphyseal dysplasia established the EDM2 locus, a region of chromosome 1 containing the COL9A2 collagen gene. We now report that affected members of this family are heterozygous for a single base transversion (T-->G) at the sixth position of the intron 3 splice donor of COL9A2. The mutation leads to skipping of exon 3 during splicing, and results in a 36-nucleotide deletion in COL9A2 transcripts derived from the mutant allele. Skipping of exon 3 predicts an in-frame deletion of 12 amino acid residues within the COL3 domain of the alpha2(IX) chain. This is the fifth instance of an exon 3 deletion within the COL3 region of collagen IX heterotrimers causing the MED phenotype, as yet the only type IX collagen defect identified in this disorder. Electron microscopy (EM) of chondrocytes obtained from articular cartilage of one affected individual in the family demonstrated normal appearing rough endoplasmic reticulum (RER). In addition, the articular cartilage matrix did not show any gross abnormalities in the quantity or caliber of collagen fibrils.

Alleles↗
PubMed2011

Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping.

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Alternative Splicing↗
PubMed1995

Systematic screening for RNA with skipped exons--splicing mutations of the ferrochelatase gene.

A systematic method was designed to screen a large population of patients with erythropoietic protoporphyria (EPP) for aberrant ferrochelatase RNA with skipped exons. The method utilizes the new junction sequence created by exon skipping as the probe to detect such RNA species. In 7 of 17 EPP families, an aberrant ferrochelatase RNA with one exon missing was observed. Two previously unreported splicing mutations were also identified in 2 EPP families. One was a G >> T transversion at the +1 position of the acceptor site of intron 8, causing exon 9 to be skipped during RNA splicing. Both the patient and her father were found to be heterozygous for this mutation. In another family, an A >> G transition at the +3 position of the donor site of intron 10 was identified, associated with exon 10 skipping during RNA splicing. Both the patient and her father were heterozygous for this mutation.

Adult↗
PubMed1998

Intron-exon structure of the MET gene and cloning of an alternatively-spliced Met isoform reveals frequent exon-skipping of a single large internal exon.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor that stimulates epithelial cell mitogenesis, motility, invasion, and morphogenesis. Its receptor is encoded by the MET proto-oncogene, a transmembrane receptor tyrosine kinase. Several studies have suggested a role for MET as a dominant oncogene in tumor development and progression. Conversely, MET is located at a region on chromosome 7q31 frequently deleted in carcinomas, suggesting that recessive mutations in MET may exist in certain cancers. To facilitate a search for mutations in MET, we have obtained the intron-exon structure of the human MET gene. We present the genomic structure of the first member of the Met receptor family to be characterized. Interestingly, MET contains a large second exon of 1214 nucleotides. We show that this exon, containing the AUG for the Met receptor, is frequently skipped in normal human tissues and cell lines, and corresponds to a ubiquitously expressed 7 kb Met transcript. This transcript yields no detectable protein product in vivo. Thus, unlike other genes, in which alternative splicing often gives rise to proteins with distinct activities, exon-skipping of MET exon 2 is predicted to decrease the abundance of a Met mRNA encoding a functional Met receptor.

Alternative Splicing↗
PubMed2005

Antisense oligonucleotides, exon skipping and the dystrophin gene transcript.

Antisense oligonucleotide induced exon skipping has recently emerged as a potential therapy to by-pass the consequences of many, but not all dystrophin mutations that lead to Duchenne muscular dystrophy. Targeted removal of one or more exons, to restore a disrupted reading frame, or omit a nonsense mutation, could lessen the consequences of an estimated 80% of dystrophin gene mutations. Promising in vitro and in vivo experiments in animal models of dystrophinopathies, as well as demonstration of induced exon skipping in cultured human myogenic cells have prompted considerable enthusiasm. Furthermore, advances in antisense oligonucleotide chemistries have resulted in the development of more stable and less toxic compounds, some of which are currently in Phase III clinical trials for selected antiviral applications. This review will summarize developments in induced exon skipping that have paved the way to clinical trials and some of the challenges and possible limitations.

Dystrophin↗
PubMed2003

Alternative splicing of the human estrogen receptor alpha primary transcript: mechanisms of exon skipping.

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.

Alternative Splicing↗
PubMed1996

Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence.

In this paper we first report that exon skipping from the dystrophin gene transcript could be induced in living cells by an antisense oligodeoxynucleotide (ODN) complementary to an exon recognition sequence (ERS). Incubation of lymphoblastoid cells with an antisense ODN against the purine-rich region of dystrophin exon 19 resulted in skipping of the exon from the dystrophin transcript. Skipping of exon 19 started to appear after 6 hours of incubation, and complete skipping was observed after 24 hours of incubation. None of the other 78 dystrophin exons were skipped, and exon 19 skipping could be induced by the sense ODN or by an antisense ODN corresponding to another ERS. These results showed that antisense ODN against ERS can induce exon skipping even in living cells and ERS is functioning as an essential cis-element for proper splicing in dystrophin pre-mRNA.

Alternative Splicing↗
PubMed1994

An exon-skipping mutation in the btk gene of a patient with X-linked agammaglobulinemia and isolated growth hormone deficiency.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease associated with a block in differentiation from pre-B to B cells. The XLA gene encodes a 659 amino acids cytoplasmic protein tyrosine kinase named btk (Bruton's tyrosine kinase). The few btk gene alterations so far reported in XLA patients are heterogenous and distributed in all domains of the btk protein. They appear to be responsible for a range of B cell immunodeficiency disorders of variable severity. Rare families in which XLA is inherited together with isolated growth hormone deficiency (IGHD) have been reported. Genetic analysis has shown that this disease association maps to the same region of the X chromosome as XLA, but whether the two phenotypes are caused by a common or different developmental or biochemical mechanism is unknown. We have analyzed the btk gene of a patient with XLA and IGHD. RT-PCR analysis of btk transcripts, sequencing data obtained from cDNA and genomic DNA and in vitro splicing assays showed that an intronic point mutation (1882 + 5G-->A) is responsible for skipping of an exon located in the tyrosine kinase domain. This exon-skipping event results in a frameshift leading to a premature stop codon 14 amino acids downstream, and in the loss of the last 61 residues of the carboxy-terminal end of the protein. Although we studied a sporadic case, the results suggest that an alteration of the btk gene might cause this unusual phenotype.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2003

Conserved sequence elements associated with exon skipping.

One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5' and 3' splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping.

Alternative Splicing↗
PubMed2003

Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Antisense oligonucleotides have been used to re-direct dystrophin pre-mRNA processing by blocking sequences crucial to pre-mRNA splicing, thereby inducing skipping of specific exons. We wished to determine which splicing motifs are most amenable as targets for antisense oligonucleotide induction of efficient and specific skipping of selected exons. METHODS: Antisense oligonucleotides were directed at regions of dystrophin exon 19 involved in pre-mRNA splicing, including the donor and acceptor splice sites and the exon splicing enhancer (ESE). Cultured myotubes were transfected with antisense oligonucleotides at various concentrations and studies undertaken to determine both specificity and efficiency of induced exon 19 skipping. RESULTS: Antisense oligonucleotides as small as 12 nucleotides targeting the ESE induced consistent and specific skipping of only exon 19 in both human and normal and mdx mouse myotubes. Antisense oligonucleotides directed at the donor and acceptor splice sites also induced specific exon 19 skipping while mismatched antisense oligonucleotides could only induce skipping when delivered at higher concentrations. No other dystrophin exons were removed from the mature mRNA as a consequence of these antisense oligonucleotides treatments. CONCLUSIONS: Antisense oligonucleotides directed at the ESE tended to be marginally more efficient than those which targeted the donor or acceptor splice sites, based on their ability to induce specific skipping at lower concentrations. The specificity of exon removal does not appear to be a function of target selection, but may reflect the combination of the splicing motifs and position of that exon in the pre-mRNA.

Animals↗
PubMed2004

Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1.

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.

Alleles↗
PubMed2004

Targeted exon skipping in transgenic hDMD mice: A model for direct preclinical screening of human-specific antisense oligonucleotides.

The therapeutic potential of frame-restoring exon skipping by antisense oligonucleotides (AONs) has recently been demonstrated in cultured muscle cells from a series of Duchenne muscular dystrophy (DMD) patients. To facilitate clinical application, in vivo studies in animal models are required to develop safe and efficient AON-delivery methods. However, since exon skipping is a sequence-specific therapy, it is desirable to target the human DMD gene directly. We therefore set up human sequence-specific exon skipping in transgenic mice carrying the full-size human gene (hDMD). We initially compared the efficiency and toxicity of intramuscular AON injections using different delivery reagents in wild-type mice. At a dose of 3.6 nmol AON and using polyethylenimine, the skipping levels accumulated up to 3% in the second week postinjection and lasted for 4 weeks. We observed a correlation of this long-term effect with the intramuscular persistence of the AON. In regenerating myofibers higher efficiencies (up to 9%) could be obtained. Finally, using the optimized protocols in hDMD mice, we were able to induce the specific skipping of human DMD exons without affecting the endogenous mouse gene. These data highlight the high sequence specificity of this therapy and present the hDMD mouse as a unique model to optimize human-specific exon skipping in vivo.

Animals↗
PubMed1994

Accelerated exon skipping of IRF-1 mRNA in human myelodysplasia/leukemia; a possible mechanism of tumor suppressor inactivation.

The transcription factor IRF-1 has been shown to function as a tumor suppressor. Here we report that a significant proportion of the IRF-1 mRNA detected in normal human hematopoietic cells and cultured cell lines lacks exon 2 (containing the AUG initiation codon) and 3 as a result of exon skipping. Surprisingly, when we examined the bone marrow and peripheral mononuclear cells from patients with myelodysplastic syndrome (MDS) or leukemia secondary to MDS, we could still detect the exon-skipped form but little or none of the intact IRF-1 mRNA. This appears to be the result of accelerated exon skipping since we could find no mutations within the exons and splicing junctions from these patients. The exon-skipped form of IRF-1 lacking exons 2 and 3 displayed neither DNA binding nor tumor suppressive activities. Thus this accelerated exon skipping may cause the inactivation of IRF-1 and thereby contribute to the development of human hematopoietic malignancies.

Cell Line↗
PubMed2006

Mutations in the SF1-U2AF59-U2AF23 complex cause exon skipping in Schizosaccharomyces pombe.

To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4+ gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4+ mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura+ mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1+, ods2+, and ods3+ encode splicing factors Prp2p/U2AF59, U2AF23, and SF1, respectively, which form a SF1-U2AF59-U2AF23 complex involved in recognition of the branch-point and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF59-U2AF23 binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF59-U2AF23 complex is essential for ordered exon joining in constitutive splicing in S. pombe.

Base Sequence↗
PubMed1997

Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis.

The cytochrome P-450 2C18 gene was found by reverse transcription-PCR to represent the most abundantly expressed gene of the P-450 2C subfamily in human epidermis. However, in addition to the canonical mRNA of nine exons, transcripts that have skipped exon 4 or 5, exons 4, 5, and 6, or exons 4, 5, 6, and 7 were also identified in this tissue. Remarkably, circular RNA transcripts synthesized by the joining of the donor and acceptor splice sites of the same exon were detected in human epidermis for exons 4 and 5. Moreover, molecules composed of exons 4, 5, and 6 with the donor splice site of exon 6 joined to the acceptor splice site of exon 4 or composed of exons 4, 5, 6, and 7 with the donor splice site of exon 7 joined to the acceptor splice site of exon 4 were also found to be present in this tissue. In rat testis, a similar analysis allowed the detection of a circular RNA molecule composed of exons 6 and 7 of the androgen binding protein (ABP) gene, with the donor splice site of exon 7 joined to the acceptor splice site of exon 6, and of an ABP mRNA which had skipped exons 6 and 7. These results apparently substantiate the hypothesis that alternative pre-mRNA splicing has the potential to generate not only mRNAs that lack one or more exons but also circular RNA molecules that are composed of the exons that are skipped. However, additional 2C18 circular species containing various combinations of exons were also detected in human epidermis, and an exon 6-skipped ABP mRNA molecule was identified in rat testis. This observation is interpreted as indicative that at low frequency, numerous circular RNA formation and exon skipping events may occur, allowing the joining of a variety of different combinations of exons. Moreover, the relative stability of these molecules is apparently the key factor that determines the relative ease of their detection.

Androgen-Binding Protein↗
PubMed1993

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

Animals↗