Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.     

Characterization of exon skipping mutants of the COP1 gene from Arabidopsis

The removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1–1 was a G→A change 4 nt upstream from the 3′ splice site of intron 5, while the mutation in cop1–2 was a G→A at the first nucleotide of intron 6, abolishing the conserved G within the 5′ splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1–8 was G→A in the final nucleotide of intron 10 abolishing the conserved G within the 3′ splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.     

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.     

Atypical 5' splice sites cause CFTR exon 9 to be vulnerable to skipping

The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3' splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3' splice sites, yet they are not skipped. Inspection of the 5' splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5' splice sites and determined the effect on exon skipping. Conversion of the upstream 5' splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5' splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5' splice site was consensus or not. These results suggested that the native downstream 5' splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5' splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5' splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5' splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5' splice sites may identify constitutive exons that are vulnerable to skipping.     

Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.     

During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.     

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.     

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.     

Exon skipping in IVD RNA processing in isovaleric acidemia caused by point mutations in the coding region of the IVD gene

Isovaleric acidemia (IVA) is a recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). We have reported elsewhere nine point mutations in the IVD gene in fibroblasts of patients with IVA, which lead to abnormalities in IVD protein processing and activity. In this report, we describe eight IVD gene mutations identified in seven IVA patients that result in abnormal splicing of IVD RNA. Four mutations in the coding region lead to aberrantly spliced mRNA species in patient fibroblasts. Three of these are amino acid altering point mutations, whereas one is a single-base insertion that leads to a shift in the reading frame of the mRNA. Two of the coding mutations strengthen pre-existing cryptic splice acceptors adjacent to the natural splice junctions and apparently interfere with exon recognition, resulting in exon skipping. This mechanism for missplicing has not been reported elsewhere. Four other mutations alter either the conserved gt or ag dinucleotide splice sites in the IVD gene. Exon skipping and cryptic splicing were confirmed by transfection of these mutations into a Cos-7 cell line model splicing system. Several of the mutations were predicted by individual information analysis to inactivate or significantly weaken adjacent donor or acceptor sites. The high frequency of splicing mutations identified in these patients is unusual, as is the finding of missplicing associated with missense mutations in exons. These results may lead to a better understanding of the phenotypic complexity of IVA, as well as provide insight into those factors important in defining intron/exon boundaries in vivo.     

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.     

Multiple features contribute to efficient constitutive splicing of an unusually large exon

Vertebrate internal exons are usually between 50 and 400 nt long; exons outside this size range may require additional exonic and/or intronic sequences to be spliced into the mature mRNA. The mouse polymeric immunoglobulin receptor gene has a 654 nt exon that is efficiently spliced into the mRNA. We have examined this exon to identify features that contribute to its efficient splicing despite its large size; a large constitutive exon has not been studied previously. We found that a strong 5′ splice site is necessary for this exon to be spliced intact, but the splice sites alone were not sufficient to efficiently splice a large exon. At least two exonic sequences and one evolutionarily conserved intronic sequence also contribute to recognition of this exon. However, these elements have redundant activities as they could only be detected in conjunction with other mutations that reduced splicing efficiency. Several mutations activated cryptic 5′ splice sites that created smaller exons. Thus, the balance between use of these potential sites and the authentic 5′ splice site must be modulated by sequences that repress or enhance use of these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon.     

Effect of 5' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.