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PubMed1998

Aberrant splicing of intron 1 leads to the heterogeneous 5' UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content.

We have previously reported that three distinct patterns of waxy (Wx) gene transcript accumulation were present in 31 rice cultivars. The cultivars with high amylose content (group I) contain a 2.3 kb mature Wx mRNA, cultivars with intermediate amylose content (group II) produce both a 3.3 kb Wx pre-mRNA, which contains intron 1, and the 2.3 kb Wx mature mRNA, and cultivars with no amylose (group III) accumulate only the 3.3 kb Wx pre-mRNA. Analyses of the cDNAs reveals that four splice donor sites and three splice acceptor sites in intron 1 give rise to six splicing patterns in 2.3 kb Wx mRNA of group II cultivars. In addition, aberrant intron 1 excision causes either deletion of 4 or 5 nucleotides, or addition of 7 and 13 nucleotides at the junction of exon 1 and exon 2 of the 2.3 kb mRNA. In contrast, only one normal splicing pattern (one splice donor site and one splice acceptor site) was found in the 2.3 kb mRNA of group I cultivars. Nucleotide sequences of the Wx intron 1 in group I and group II cultivars differ by 16 individual bases. We suggest that these deletions or additions contribute to inefficient splicing of intron 1 from the 3.3 kb Wx pre-mRNA, as well as an aberrant splicing of the Wx intron 1 to produce the 2.3 kb mRNA with a heterogeneous 5' untranslated region (5'-UTR). As a consequence, the total amount of translatable Wx mRNA, and therefore the Wx protein and amylose content, are reduced in the group II cultivars compared with the group I cultivars.

Alternative Splicing↗
PubMed1999

Supravalvular aortic stenosis: a splice site mutation within the elastin gene results in reduced expression of two aberrantly spliced transcripts.

We have screened the elastin gene for mutations responsible for supravalvular aortic stenosis (SVAS) in two large, independently collected families with isolated (nonsyndromic) SVAS. By single-strand conformation polymorphism and heteroduplex analysis, we have identified a C to G transversion within the acceptor splice site of exon 16 in SVAS patients from both families. This mutation segregates in both families with high penetrance of SVAS, and all affected individuals carry the mutation. Haplotype analysis by using closely linked polymorphisms, including a previously unreported BfaI restriction fragment length polymorphism within the 3'-UTR of the elastin gene, indicates that the mutations found in the two apparently non-overlapping kindreds are identical by descent. To study the effect of the mutation on the expression of the mutant allele, we have established a primary skin fibroblast culture from one of the affected individuals. Reverse transcription/polymerase chain reaction analysis of elastin mRNA species indicates that the mutation results in two abnormal elastin mRNA species. One mutant elastin mRNA is generated by the activation of a cryptic splice site that lies within intron 15 and that adds 44 bp of intronic sequence to the sequence encoded by exon 16. This insertion creates a frame shift that results in a 59-amino-acid-long abnormal protein sequence and leads to a termination codon in the mRNA sequence encoded by exon 17. The smaller abnormal mRNA species arises as a consequence of the skipping of exon 16. This study demonstrates, for the first time, the expression of mutant alleles of the elastin gene in patients with isolated SVAS.

Alternative Splicing↗
PubMed1992

Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice.

The serum level of the fourth component of complement (C4) in mice bearing the H-2k haplotype is only 1/10 to 1/20 of that of non-H-2k mice. We have analyzed C4 cDNA clones from B10.BR(H-2k) mouse liver and found aberrant C4 cDNA which contained a 200-base pair (bp) insertion between the exon 13 and exon 14 encoded sequences in addition to the normal C4 cDNA. The 5' 148 bp and the 3' 52 bp of this insert were derived from the B2 sequence, the short interspersed repeats of mouse genome, and the central part of intron 13, respectively. Sequence analysis of intron 13 of the C4k gene showed the presence of a complete copy of a B2 consensus sequence. The structure of aberrant C4 mRNA indicated that the possible 3' splice site in the B2 sequence and the cryptic 5' splice site in intron 13 were used. Both the insertion of the B2 sequence into intron 13 and the presence of aberrant mRNA in the liver were specific to H-2k-bearing mice, suggesting that the aberrant splicing due to the B2 insertion is the basis for low C4 expression in H-2k mice.

Animals↗
PubMed1994

A hereditary C3 deficiency due to aberrant splicing of exon 10.

Hereditary C3 deficiency in a 22-year-old woman was studied. Previous works indicated that C3 could not be detected in the serum of such a patient by enzyme immunoassay. In this study, we demonstrated that C3 genes of this patient and her parents have no gross structural aberration. However, C3 mRNA was almost not detectable in the skin fibroblasts of this patient. The activity of the patient's C3 upstream regulatory elements was tested and showed no functional abnormality. Using reverse-transcriptase polymerase chain reaction (RT-PCR) to amplify RNA from LPS-stimulated patient's fibroblasts, two shorter cDNAs within C3 exon 8 to exon 12 were noted. DNA sequence analysis of the RT-PCR products revealed that one deleted 116 nucleotides of the full exon 10 and the other deleted 34 nucleotides in the 3' region of exon 10. A single base substitution (G to T) in the splice donor site of intron 10 was identified. This aberrant splicing involving exon 10 could result in translational pretermination at exon 11. Thus, this mutation provided the molecular basis for the deficiency of C3 in the patient.

Adult↗
PubMed2006

BCR-ABL1 induces aberrant splicing of IKAROS and lineage infidelity in pre-B lymphoblastic leukemia cells.

Pre-B lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement exhibit an undifferentiated phenotype. Comparing the genome-wide gene expression profiles of normal B-cell subsets and BCR-ABL1+ pre-B lymphoblastic leukemia cells by SAGE, the leukemia cells show loss of B lymphoid identity and aberrant expression of myeloid lineage-specific molecules. Consistent with this, BCR-ABL1+ pre-B lymphoblastic leukemia cells exhibit defective expression of IKAROS, a transcription factor needed for early lymphoid lineage commitment. As shown by inducible expression of BCR-ABL1 in human and murine B-cell precursor cell lines, BCR-ABL1 induces the expression of a dominant-negative IKAROS splice variant, termed IK6. Comparing matched leukemia sample pairs from patients before and during therapy with the BCR-ABL1 kinase inhibitor STI571 (Imatinib), inhibition of BCR-ABL1 partially corrected aberrant expression of IK6 and lineage infidelity of the leukemia cells. To elucidate the contribution of IK6 to lineage infidelity in BCR-ABL1+ cell lines, IK6 expression was silenced by RNA interference. Upon inhibition of IK6, BCR-ABL1+ leukemia cells partially restored B lymphoid lineage commitment. Therefore, we propose that BCR-ABL1 induces aberrant splicing of IKAROS, which interferes with lineage identity and differentiation of pre-B lymphoblastic leukemia cells.

Alternative Splicing↗
PubMed1999

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.

Acetylcholinesterase↗
PubMed2005

Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene.

Low FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F 7 gene:a G to A transition in the 5' donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2-4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Delta 2, 3), leading to an in-frame deletion of the propeptide and gamma-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Delta 2,3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient's phenotype.

Base Sequence↗
PubMed2000

Aberrant splicing of tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism linked to chromosome 17.

Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.

Adult↗
PubMed2002

Aberrant splicing of FHIT transcripts in human gastric cancer cell lines.

Alterations of the FHIT gene occur as frequent events in several human cancers. Replacement of exogenous wild-type FHIT gene has been shown to induce suppression of tumorigenicity of human FHIT-negative cells in nude mice and aberrant FHIT transcripts have been observed in a variety of human solid tumors. In the presence study, we performed a nested reverse transcription-polymerase chain reaction (RT-PCR) analysis to identify aberrant FHIT transcripts in 6 gastric cancer cell lines. In addition to the wild-type FHIT transcript, small-sized transcripts with various number and lengths were observed in all of the cell lines examined. Sequence analysis confirmed that different types of truncated transcripts included exonic deletions, insertions of intron 5 sequences between exons, and combinations of both. Most of these transcripts lacked exon 5 in which translation initiation codon is located. Aberrant transcripts with partial exonic deletions, resulting from activation of cryptic splice sites, were also observed in 5 cell lines. Additionally, multi-step splice patterns, indicative of additional downstream processing, were observed in several cancer lines. Our results suggest that the aberrant FHIT transcripts in gastric cancer cell lines resulted from faulty splicing, including exon skipping, selection of cryptic splice site and additional downstream splice processing.

Acid Anhydride Hydrolases↗
PubMed1996

Retarded and aberrant splicings caused by single exon mutation in a phosphoglycerate kinase variant.

The molecular abnormality of a phosphoglycerate kinase variant which was associated with severe tissue enzyme deficiency and episodes of muscle contractions and myoglobinuria was examined. Analysis of the patient's DNA showed the existence of a nucleotide transversion A/T - C/G in exon 7. No other nucleotide change was detected in the coding region of the variant gene. The mutation should produce a single amino acid substitution Glu - Ala at protein position 251 counting from the NH2-terminal acetyl serine residue. The protein abnormality caused by the amino acid substitution cannot explain the enzyme deficiency. Northern blot hybridization indicated that the PGK mRNA content of the patient's lymphoblastoid cells was only about 10% of that of normal. Nucleotide sequence analysis revealed the existence of two PGK mRNA components in the patient's cells. The major component corresponds to the normal PGK mRNA except for A - C change at nucleotide position 755 counting from adenine of the chain initiation codon. The minor component contains 5' region (52 bases) of intron 7 between exon 7 and exon 8. An inframe chain termination codon exists in the minor mRNA component, and the COOH-terminal half is expected to be deleted in the translation product. These results indicate that the low PGK activity in the patient's tissues is mainly due to retarded and aberrant pre-mRNA splicings caused by the change of the consensus 5' splice sequence AGgt to a nonconsensus sequence CGgt at the junction between exon 7 and intron 7 of the variant gene.

Adolescent↗
PubMed2000

Identification and elimination of an aberrant splice product from cDNAs encoding the human adenovirus type 5 E4orf6 protein.

Growing awareness of the central role of the E4orf6 protein in regulating the infectious cycle of human adenoviruses has led to greatly intensified efforts to define its functions and mechanisms of action. Many workers employ cDNAs to generate plasmid or viral vectors to express E4orf6 in the absence of other viral products. In addition to the normal 34-kDa product, such vectors consistently produce a polypeptide of about 8 kDa. In the present report we show that this protein is produced by an aberrant mRNA utilizing the 5' splice donor site used normally by the virus to produce the E4orf6/7 product, which shares 58 residues with E4orf6. This amino terminal coding sequence is linked to a 3' sequence via a novel splice acceptor site in an alternative reading frame of the E4orf6 cDNA. The 5' donor site was altered by PCR-directed mutagenesis to yield a construct that produces high levels of E4orf6 in the absence of the 8-kDa polypeptide. This construct should eliminate some of the problems encountered previously using the wild-type E4orf6 coding sequence.

Adenovirus E4 Proteins↗
PubMed1994

Multiple aberrant splicing of the p53 transcript without genomic mutations around exon-intron junctions in a case of chronic myelogenous leukaemia in blast crisis: a possible novel mechanism of p53 inactivation.

We found three truncated p53 transcripts in a patient with chronic myelogenous leukaemia in blast crisis carrying chromosome 17 abnormalities. Sequencing of these transcripts revealed complete absence of the entire exons 7, 8 and 9 in one, exons 8 and 9 in another, and exon 10 in the other. Sequencing analysis of genomic DNA, however, revealed no mutation in exons 6-10 and their flanking introns. These results suggest that the aberrant p53 transcripts in this case might not result from splicing mutations but from an unknown affected splicing process.

Base Sequence↗
PubMed1996

Aberrant splicing and truncated-protein expression due to a newly identified XPA gene mutation.

A group A xeroderma pigmentosum (XPA) patient, XP2NI, is a compound heterozygote with a newly identified G to C transversion at the last nucleotide in exon 5 in one chromosome, and with the known splicing mutation in intron 3 in another chromosome in the XPA gene. XP2NI had mild skin symptoms and the cells were slightly less sensitive to UV radiation than the cells of typical severe XPA patients who have the splicing mutation in intron 3 homozygously. Reverse transcriptase (RT)-PCR and sequencing of the PCR products revealed that the mutation in exon 5 resulted in producing three types of aberrant mRNA, lacking 7 nucleotides at the end of exon 5, lacking entire exon 5, and lacking exons 3, 4 and 5. A significant amount of a truncated type of protein was produced in XP2NI cells, and the size of the protein indicated that it should have been translated from the mRNA, lacking the 7 nucleotides and retained one of the zinc-finger domains required for the DNA repair activity. The clinical mildness of XP2NI may be due to the residual DNA repair activity of the truncated XPA protein, while no XPA protein was detected in the XPA cells with the homozygous intron 3 splicing mutation.

Base Sequence↗
PubMed2002

A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome.

Human Pex16p, a peroxisomal membrane protein composed of 336 amino acids, plays a central role in peroxisomal membrane biogenesis. A nonsense mutation (R176ter) in the PEX16 gene has been reported in the case of only one patient (D-01) belonging to complementation group D of the peroxisome biogenesis disorders. We have now identified two patients belonging to group D (D-02 and D-03) whose fibroblasts were found to contain no peroxisomal membrane structure ghosts. Molecular analysis of the PEX16 gene revealed aberrant cDNA species lacking 65 bp, corresponding to exon 10 skipping caused by a splice site mutation (IVS10 + 2T -->C). Both patients, although unrelated, were homozygous for this mutation. This mutation changes the amino acid sequence starting from codon 298 and introduces a termination codon at codon 336. As a consequence, the cell's ability to membrane synthesis and protein import is disrupted, which implies that the changed C terminus of the Pex16p in these patients likely affects its function.

ATP-Binding Cassette Transporters↗
PubMed1989

Leukocyte adhesion deficiency. Aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype.

Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63% and 53% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5' splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype.

Amino Acid Sequence↗
PubMed1999

Expression of aberrantly spliced oncogenic ikaros isoforms in childhood acute lymphoblastic leukemia.

PURPOSE: We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children. PATIENTS AND METHODS: We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver-derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations. RESULTS: In each of the ALL cases, we found high-level expression of a non-DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver-derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non-DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition. CONCLUSION: Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non-DNA-binding Ikaros isoforms that are reminiscent of the non-DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.

Adolescent↗
PubMed2000

A branch site mutation leading to aberrant splicing of the human tyrosine hydroxylase gene in a child with a severe extrapyramidal movement disorder.

We report a branch site mutation in the gene of the enzyme tyrosine hydroxylase (TH): a -24t > a substitution two bases upstream of the adenosine in the branchpoint sequence (BPS) of intron 11. As normal lariat formation is therefore prevented, alternative splicing takes place; use of the BPS of intron 12 results in skipping of exon 12, whereas the use of a cryptic branch site in intron 11 leads to partial retention of this intron in the mRNA. This leads in both cases to an aberrant protein product. In the one case, skipping of exon 12 results in the absence of 32 amino acids. In the other, retention of 36 nucleotides of intron 11 in the mRNA results in the incorporation of twelve additional amino acids. The functional consequences of this mutation for the patient, who is also heterozygous for another previously identified mutation, become apparent in a severe clinical phenotype.

Basal Ganglia Diseases↗
PubMed1995

Expression of aberrantly spliced growth hormone receptor mRNA in the sex-linked dwarf chicken, Gifu 20.

The structure and expression of the growth hormone receptor (GHR) gene have been studied in a sex-linked dwarf (SLD) chicken strain, Gifu 20. No gross structural change in the receptor gene was observed by Southern blot analysis, however, a transcript of 5.5 killobases (kb), which was approximately 1 kb larger than the usual chicken full length-GHR mRNA was detected in the dwarf chicken by Northern blot analysis. GHR cDNA was obtained from dwarf chicken liver RNA by reverse transcription-polymerase chain reaction, and nucleotide sequence analysis revealed that it contained an intron sequence of the GHR gene. The unspliced intron showed a single point mutation at the donor site from GT to GC, and an aberrant stop codon was generated in the extracellular domain-coding region. Thus, the mutation resulting in an inappropriate splicing of the GHR gene transcript is considered to be the cause of this dwarf chicken.

Alternative Splicing↗