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Results for “cryptic splice site”

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At least 19 recordsLinked to original sources

PubMed2003

Intrinsic differences between authentic and cryptic 5' splice sites.

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5' splice sites (5'ss), we systematically analyzed a set of 76 cryptic 5'ss derived from 46 human genes. These cryptic 5'ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5'ss. Statistical analysis of the strengths of the 5'ss using the Shapiro and Senapathy matrix revealed that authentic 5'ss have significantly higher score values than cryptic 5'ss, which in turn have higher values than the mutant ones. beta-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the beta-globin authentic and cryptic 5'ss, but not their surrounding context, determine the correct 5'ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5'ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5'ss-disrupting mutations at the mRNA level.

Alternative Splicing↗

PubMed1999

A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation.

DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5' terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One activated donor site, located nine nt 5' to the normal splice donor site, begins with the dinucleotide GC. While non-consensus, this sequence still permits both trans-esterification reactions of pre-mRNA splicing. A second cryptic site located 23 nt 5' to the normal splice site and beginning with GA, undergoes the first trans-esterification reaction leading to lariat formation, but lacks the ability to participate in the second reaction. Accumulation of this splicing intermediate and use of an innovative reverse transcriptase-polymerase chain reaction technique (J. Vogel, R.H. Wolfgang, T. Borner [1997] Nucleic Acids Res 25: 2030-2031) led to the identification of 3' intron sequences needed for lariat formation. In most splicing reactions, neither cryptic site is recognized. Most mature transcripts include intron 3, while the second most frequent class lacks exon 3. Traditionally, the former class of transcripts is taken as evidence for the intron definition of splicing, while the latter class has given credence to the exon definition of splicing.

Alternative Splicing↗

PubMed1999

Cryptic intron activation within the large exon of the mouse polymeric immunoglobulin receptor gene: cryptic splice sites correspond to protein domain boundaries.

The fourth exon of the mouse polymeric immuno-globulin receptor (pIgR) is 654 nt long and, despite being surrounded by large introns, is constitutively spliced into the mRNA. Deletion of an 84 nt sequence from this exon strongly activated both cryptic 5' and 3' splice sites surrounding a 78 nt cryptic intron. The 84 nt deletion is just upstream of the cryptic 3' splice site; the cryptic 3' splice site was likely activated because the deletion created a better 3' splice site. However, the cryptic 5' splice site was also required to activate the cryptic splice reaction; point mutations in either of the cryptic splice sites that decreased their match to the consensus splice site sequence inactivated the cryptic splice reaction. The activation and inactivation of these cryptic splice sites as a pair suggests that they are being co-recognized by the splicing machinery. Interestingly, the large fourth exon of the pIgR gene encodes two immunoglobulin-like extracellular protein domains; the cryptic 3' splice site coincides with the junction between these protein domains. The cryptic 5' splice site is located between protein subdomains where an intron is found in another gene of the immunoglobulin superfamily.

Animals↗

PubMed1990

Mechanism for cryptic splice site activation during pre-mRNA splicing.

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby "cryptic" 5' splice sites. Here we show that the effects of single-base substitutions within a 5' splice site can be completely or partially suppressed by cis mutations that improve the overall complementarity of the site to U1 snRNA. We further show that in the presence of the normal 5' splice site, a cryptic 5' splice site can be activated by increasing its complementarity to U1 snRNA. U1 snRNP binding experiments confirm that cryptic 5' splice sites are activated when their affinity for U1 snRNP approaches that of the authentic 5' splice site. Based upon these results, we propose a spliceosome competition model for 5'-splice-site selection and cryptic 5'-splice-site activation. We discuss our results with regard to the factors involved in 5'-splice-site recognition.

Animals↗

PubMed2001

Activation of a cryptic splice site of PTEN and loss of heterozygosity in benign skin lesions in Cowden disease.

Cowden disease is an autosomal dominant syndrome characterized by facial trichilemmomas, acral keratoses, papillomatous papules, mucosal lesions, and an increased risk for breast and nonmedullary thyroid cancer. Here, we describe a novel PTEN splicing site mutation in a family with classical Cowden disease and we studied benign skin lesions typical for Cowden disease for loss of heterozygosity. We found a PTEN IVS2 + 1G > Alpha 5'-splicing acceptor mutation resulting in activation of a cryptic splice site. Activation of this cryptic splice site is predicted to result in a frameshift with a premature stop codon, thus disrupting the phosphatase core motif of PTEN. Loss of heterozygosity analysis of two trichilemmomas, one fibroma, and three acanthomas of the index patient demonstrated loss of heterozygosity at the PTEN locus in four of these lesions. In conclusion, our data demonstrate that a PTEN splicing site mutation causes activation of a cryptic splice site, which results in aberrant transcripts.

Aged↗

PubMed2005

Mutation of acceptor splice site of the SEDL gene in X-linked spondyloepiphyseal dysplasia tarda causes the activation of cryptic splice site.

OBJECTIVE: To further investigate the genetic basis of hereditary X-linked spondyloepiphyseal dysplasia tarda (SEDL) and provide useful information for the prevention and treatment of the disease. METHODS: RT-PCR and cDNA sequencing were used to test mRNA expression of SEDL gene in a patient with 13 bp deletion of SEDL gene involving the acceptor splice site of intron 5. RESULTS: Of two different sizes of mRNA products identified in the patient, the 393 bp product was created due to the activation of cryptic splice site within exon 6; the 433 bp product was completely consistent with the part of genomic sequence on chromosome 8. CONCLUSION: The intragenic deletion that occurred in the acceptor splice site of the 3'region of intron 5 and the 5' coding region of exon 6 results in the activation of a cryptic splice site within exon 6, which causes 47 bp deletion of the resulting mRNA followed by a frameshift that would add two missense amino acids and then be followed by a termination codon (D109-S123del; S124fsX126). In addition, the mutation may activate the transcription of pseudogene SEDLP2 on chromosome 8 to partly complement the function of SEDL protein.

Adolescent↗

PubMed2004

Exon junction sequences as cryptic splice sites: implications for intron origin.

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.

Actins↗

PubMed1995

An aberrant splicing using a 3' cryptic splice site within the CH1 exon induces truncated mu-chain production.

AT8--1-12--5-1, an Abelson virus-transformed immature B-cell line, produced truncated mu-chains. Sequencing analysis of the mu-expressed allele revealed that the variable region was an out-of-frame VH7183-DSP2-JH3 complex. Two cDNA clones (5-1 cDNA1 and 5-1 cDNA2) derived from the transcripts of the mu-expressed allele were cloned and sequenced. Sequencing analysis of 5-1 cDNA1 revealed that the VH7183-DSP2-JH3 sequence jointed to the CH1 exon at 136 bp, 3' from the 5' end of the CH1 exon, resulting in the change of the reading frame from out-of-frame to in-frame. On the other hand, sequencing analysis of 5-1 cDNA2, which appeared to have derived from intron-containing premature mRNA, revealed that the J-C intron sequence joined to the CH1 exon at 110 bp 3' from the 5' end of the CH1 exon, indicating the deletion of 109 bp including the 3' splice site of the CH1 exon. These results demonstrate that the deletion of the authentic 3' splice site of the CH1 exon induced activation of the cryptic splice site within the CH1 exon. This was followed by splicing of the variable region to the CH1 exon at the cryptic splice site at 136 bp 3' from the 5' end of the CH1 exon, resulting in the change of the reading frame from out-of-frame to in-frame, followed by the truncated mu-chain production.

Alleles↗

PubMed2004

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5' splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5' splice site to several cryptic, noncanonical 3' splice sites immediately adjacent to the normal 3' splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3' splice site sequences to pair to a given 5' splice site, albeit with tight constraints for maintaining the 3' splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.

AMP-Activated Protein Kinase Kinases↗

PubMed1992

A base substitution at the splice acceptor site of intron 5 of the COL1A2 gene activates a cryptic splice site within exon 6 and generates abnormal type I procollagen in a patient with Ehlers-Danlos syndrome type VII.

The dermal type I collagen of a patient with Ehlers-Danlos type VIIB (EDS-VIIB) contained normal alpha 2(I) chains and mutant pN-alpha 2(I)' chains in which the amino-terminal propeptide (N-propeptide) remained attached to the alpha 2(I) chain. Similar alpha 2(I) chains were produced by cultured dermal fibroblasts. Amino acid sequencing of tryptic peptides, prepared from the mutant amino-terminal pN-alpha 2(I) CB1' peptide, indicated that five amino acids, including the N-proteinase (the specific proteinase that cleaves the procollagen N-propeptide) cleavage site, had been deleted from the junction of the N-propeptide and the N-telopeptide (the nonhelical domain at the amino-terminus of the alpha chains of fully processed type I polypeptide chains) of the mutant pro-alpha 2(I)' chain. The corresponding 15 nucleotides, which were deleted from approximately half of the alpha 2(I) cDNA polymerase chain reaction products, of the alpha 2(I) cDNA polymerase chain reaction products, were encoded by the +1 to +15 nucleotides of exon 6 of the normal alpha 2(I) gene (COL1A2). These 15 nucleotides were deleted in the splicing of alpha 2(I) pre-mRNA to mRNA as a result of inactivation of the 3' splice site of intron 5 by an AG to AC mutation and the activation of a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6. Loss of the N-proteinase cleavage site explained the persistence of the pN-alpha 2(I)' chains in the dermis and in fibroblast cultures. Collagen production by cultured dermal fibroblasts was doubled, possibly due to reduced feedback inhibition by the N-propeptides. In contrast to previously reported cases of EDS-VIIB, Lys5 of the N-telopeptide was not deleted and appeared to take part in the formation of intramolecular cross-linkages. However, increased collagen solubility and abnormal extraction profiles of the mutant type I collagen molecules indicated that collagen cross-linking was abnormal in the dermis. The proband and her son were heterozygous for the mutation. It is likely that the heterozygous loss of the N-proteinase cleavage site, with persistence of a shortened N-propeptide, was the major factor responsible for the EDS-VIIB phenotype.

Adult↗

PubMed2006

Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon.

In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

Base Sequence↗

PubMed2003

Transcobalamin deficiency due to activation of an intra exonic cryptic splice site.

Transcobalamin (TC), a vitamin B12 (cobalamin, Cbl) binding protein in plasma, promotes the cellular uptake of the vitamin by receptor-mediated endocytosis. Inherited TC deficiency is an autosomal recessive disorder characterized by megaloblastic anaemia caused by cellular vitamin B12 depletion. It may be accompanied by neurological complications, including a delay in psychomotor and mental development. This report describes three sisters with inherited TC deficiency resulting from a splicing defect in the TC gene. A point mutation was identified in intron 3 splice site of the TC gene that activates a cryptic splice site in exon 3. The transcript generated has an in-frame deletion of 81 nucleotides and the resulting truncated protein is unstable and not secreted by the cells. Until now, genetic studies have been reported in only five patients with TC deficiency and the molecular defect was different in each of them, which gives evidence for a genetic heterogeneity of the disease.

Adult↗

PubMed1996

Expression of the Volvox gene encoding nitrate reductase: mutation-dependent activation of cryptic splice sites and intron-enhanced gene expression from a cDNA.

Use of the nitrate reductase encoding gene (nitA) as selection marker has facilitated the successful nuclear transformation of Volvox carteri. The Volvox nitA gene contains 10 introns. A stable nitA mutation in the Volvox recipient strain 153-81 resides in a G-to-A transition of the first nucleotide in the 5' splice site of nitA intron 2. This mutation resulted in at least three non-functional splice variants, namely: (1) intron 2 was not spliced at all; (2) a cryptic 5' splice site 60 nt upstream or (3) a cryptic 5' splice site 16 nt downstream of the mutation were activated and used for splicing. When we used nitA cDNA (pVcNR13) for transformation of V. carteri 153-81, a low efficiency of about 5 x 10(-5) transformants per reproductive cell was observed. Re-integration of either intron 1 (pVcNR15) or introns 9 and 10 (pVcNR16) in the transforming cDNA increased transformation rates to 5 x 10(-4). In parallel, pVcNR15-transformed Volvox exhibited growth rates that were 100-fold increased over the pVcNR13-transformed alga. This intron-enhancement of nitA gene expression appears to be associated with post-transcriptional processing and 'channelling' of the message. These data suggest an important role of splicing for gene expression in V. carteri.

Base Sequence↗

PubMed2000

Cryptic splice site activation during RNA processing of MLL/AF4 chimeric transcripts in infants with t(4;11) positive ALL.

Co-expression of multiple variants of the MLL/AF4 fusion transcript is a common phenomenon in patients with acute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23). Different transcriptional and post-transcriptional mechanisms were found to contribute to the heterogeneity of the chimeric transcripts. Multiple splice variants are generated by utilizing alternative splice sites that result in the joining of different MLL-exons within the breakpoint cluster region to one of three exons in the AF4 fusion partner. To address the question of how splice site selection occurs during RNA processing, we investigated der(11) transcripts in 10 infants with t(4;11) positive ALL. Specific RT-PCR products were analyzed by Southern blot hybridization, SSCP, endonuclease digestion, cloning and sequencing. In patients co-expressing as many as six different chimeric mRNA species, activation of cryptic splice sites has been detected in MLL-exons 8 and 10. This led to the formation of four novel transcript variants, three of which maintained open reading frames (ORFs). Patients with cryptic donor site activation in MLL-exon 8 did not have any MLL-exon 8/AF4 transcripts using the authentic 5' splice site, although this site is 100% homologous to the consensus sequence. However, since MLL-exon 8 does not end in-phase, the use of the authentic splice site would result in loss of the ORF of the fusion message. The activated cryptic splicing sites are located in the vicinity of the polypurine stretches present in MLL-exons 8 and 10, which are known to function as splicing enhancers recognized by SR proteins. We postulate that both the nonsense-mediated decay eliminating correctly spliced MLL-exon 8/AF4 mRNAs and activation of suboptimal splicing sites contribute to the diversity of MLL/AF4 RNA species.

Alternative Splicing↗

PubMed2006

Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease.

We describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534-3C > A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon in VWF propeptide, so the proband VWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534-3C > A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3 VWD. This mutation therefore appears to be peculiar to type 1 VWD.

Adenine↗

PubMed1998

Implications of a novel cryptic splice site in the BRCA1 gene.

This study was designed to determine the significance of a single intronic base change (IVS5-12 G-->A) found in a family with a history of breast cancer. This change is predicted to form a cryptic splice site resulting in the addition of 11 nucleotides to the BRCA1 transcript. The BRCA1 gene of the relatives and control individuals was sequenced and analyzed using RT-PCR, ASO hybridization, and size fractionation. All patients showed an 11 nucleotide insert at the intron 5/exon 6 boundary. This variant is likely to form a short protein product incapable of the hypothesized tumor suppressor functions of the BRCA1 gene. This information is important for providing counseling for families with this cryptic splice site and a family history of breast cancer.

Base Sequence↗

PubMed1998

Usage of cryptic splice sites in citrullinemia fibroblasts suggests role of polyadenylation in splice-site selection during terminal exon definition.

Citrullinemia is a human genetic disease caused by a deficient argininosuccinate synthetase. In fibroblasts established from a citrullinemia patient with a mutation at the 3' splice site of the terminal intron of the gene, three cryptic 3' splice sites; i.e., SA1275, SA1636, and SA1663, residing on the terminal exon were activated. The usage of the cryptic sites showed a gradient, with the most downstream site having the highest usage; i.e., SA1663 > SA1636 > SA1275. However, when these cryptic sites were relocated to the internal exon, SA1636 was used the most. The splice-site strength of SA1636 was at least 10-fold higher than that of SA1663 in this situation. The results suggest that the preferential usage of SA1663 residing on the terminal exon may depend on its proximity to the poly(A) signal rather than on the strength of the splice site. Furthermore, when the strength of the downstream-most splice site increased, almost all the RNAs spliced to this site. However, in the presence of the wild-type splice site, all the RNAs were processed to the authentic site. Apparently, the selection of splice site can be revealed only when the sites being selected do not differ too much in their strength. By using a naturally occurring human mutant gene as a model, this study reveals that polyadenylation may play an important role in the selection of splice site during terminal exon definition.

Amino Acid Metabolism, Inborn Errors↗

PubMed1989

Mutation of 3' splice sites in two different class I genes results in different usage of cryptic splice sites.

To determine the pattern of alternative splicing at the 5' end of class I genes, the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes were mutated from AG to GG (H-2Dd) or CG (H-2Kd). The mutant genes were transfected into L cells, and RNA from clones expressing these Ag was used for analysis by RNase and S1 nuclease mapping techniques. The first intervening sequence of both class I genes contains several potential 3' splice acceptor sites. However, a clear preference for only one site was detected in each of the H-2Dd and H-2Kd mRNA. Examination of the endogenous H-Dd and H-2Kd class I transcripts in normal murine tissues and in tumors demonstrated that the alternatively spliced mRNAs were produced, but at a low frequency. Infection of transfected L cells or tumor lines with vesicular stomatitis virus altered the level of differentially spliced message in these cells.

Animals↗