Functional Splicing Assay Research Articles

A complex splicing defect associated with homozygous ankyrin-deficient hereditary spherocytosis

AbstractDefects in erythrocyte ankyrin are the most common cause of typical, dominant hereditary spherocytosis (HS). Detection of ankyrin gene mutations has been complicated by allelic heterogeneity, large gene size, frequent de novo mutations, and associated mRNA instability. Using denaturing high-performance liquid chromatography (DHPLC)–based mutation detection, a mutation in the splice acceptor of exon 17 was discovered in a Turkish family. Reticulocyte RNA and functional minigene splicing assays in heterologous cells revealed that this mutation was associated with a complex pattern of aberrant splicing, suggesting that removal of intron 16 is important for ordered ankyrin mRNA splicing. As predicted by clinical, laboratory, and biochemical studies, the parents were heterozygous and the proband was homozygous for this mutation. These data indicate that DHPLC offers a highly sensitive, economic, and rapid method for mutation detection and, unlike previously suggested, homozygosity for a mutation associated with dominant ankyrin-linked HS may be compatible with life.

Argininosuccinate lyase deficiency: mutational spectrum in Italian patients and identification of a novelASLpseudogene

Argininosuccinic aciduria (ASAuria) is an inborn error of metabolism caused by mutations in the argininosuccinate lyase (ASL) gene, which leads to the accumulation of argininosuccinic acid (ASA) in body fluids and severe hyperammonemia. A severe neonatal form and a milder late-onset variant are described. We report a novel ASL pseudogene located in the centromeric region of chromosome 7, 14 novel mutations in the ASL gene, and a novel intronic polymorphism found in a cohort of Italian patients. Our approach relied exclusively on genomic DNA analysis. We found seven missense mutations, two nonsense, three small insertions/deletions, and two splicing mutations. Only two patients harbored previously described mutations, and among the novel variants only two were present in more than one kindred. The pathogenicity of the splicing mutations was demonstrated by a functional splicing assay that employed a hybrid minigene. We also performed molecular modeling using the reported three-dimensional structure of ASL to predict the functional consequences of the missense mutations. There was no genotype-phenotype correlation. Patients with neonatal onset display developmental delay and seizures despite adequate metabolic control. Moreover, hepatomegaly, fibrosis, and abnormal liver function tests are common complications in these patients, but not in patients with the late infancy form. We stress the importance of mutation analysis in patients with ASAuria, to confirm the clinical diagnosis, and to perform DNA-based prenatal diagnosis in future pregnancies of these families.

Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization

Genomic variations with no apparent effect ("neutral polymorphisms") may have a significant effect on splicing. The effect of this type of mutation is difficult to spot, unless a functional assay is undertaken. In our study, DNA sequencing of a patient with clinically defined neurofibromatosis type 1 (NF1) showed only a single polymorphism in intron 30 due to an A>G transition 279 nucleotides from the 3' splice site. Using a minigene splicing assay we conclusively show that this change produces a cryptic exon with a 3' SS defined by the nucleotide change and the unexpected activation of a very weak 5'SS. Further site directed mutagenesis studies aimed at identifying the signals involved in the cryptic exon inclusion were carried out. Interestingly we find that particular characteristics of the cryptic 5' SS are essential for its inclusion. Significantly an additional single nucleotide change disrupting the cryptic 5'ss consensus sequence rescues the effect of the pathogenetic mutation resulting in normal splicing.

Characterization of two novel GBA mutations causing Gaucher disease that lead to aberrant RNA species by using functional splicing assays

The correct identification of disease-causing mutations from the background of harmless nucleotide polymorphisms/substitutions has become a critical issue in the investigation of human genetic diseases. Here, we describe two novel disease-causing splicing mutations in the glucocerebrosidase gene, g.4252C>G and g.4426A>G, that have been found in two patients affected by Gaucher disease. The g.4252C>G substitution occurred in intron 5 and was located 12 nucleotides upstream of exon 6 acceptor site whilst the g.4426A>G mutation was located within this exon, 12 nucleotides upstream of the donor site. An in silico analysis suggested that both mutations could have altered the splicing process of exon 6 by creating a novel acceptor and donor site, respectively. However, because the wild-type acceptor and donor sites of exon 6 were not apparently affected, the severity of both mutations could not be established by simple sequence analysis alone. Nonetheless, the use of minigene functional assays to complement transcript analysis of patient fibroblasts shows that both mutations cause the almost complete switch of splice site usage from the wild-type to the newly-created ones, thus providing a functional explanation for the appearance of disease.

An Intron Splicing Enhancer Element, UGCAUG, Is Evolutionarily Conserved near Erythroid Protein 4.1R Exon 16 and Other Tissue-Specific Alternative Exons.

Alternative pre-mRNA splicing switches provide an important mechanism for regulating gene expression during differentiation. In differentiating erythroblasts, stage-specific activation of protein 4.1R exon 16 splicing promotes the synthesis of protein isoforms with high affinity for spectrin and actin, which is important for mechanical stabilization of the red cell membrane skeleton. Regulation of this alternative splicing switch is mediated by the interaction of multiple trans-acting splicing factor proteins with cis RNA elements in the pre-mRNA. We previously identified an intron splicing enhancer downstream of exon 16 that is essential for optimal inclusion of exon 16 in functional splicing assays. This enhancer contains three repeats of the sequence UGCAUG, a highly specific binding site for the newly described Fox-1 family of splicing activator proteins. Our results implicate the highly homologous Fox-2 protein as a candidate splicing activator for exon 16 inclusion during erythroid differentiation, based on the observations that Fox-2 is expressed in mouse erythroblasts; and that it both binds to the intron 16 enhancer, and enhances exon 16 splicing, in a UGCAUG-dependent manner. Here we also report genome sequence analyses showing that UGCAUG is evolutionarily conserved in the proximal downstream intron sequence near many tissue-specific alternative exons. First, examination of the 4.1R genes revealed that three repeats of the UGCAUG motif were conserved in introns of all mammals tested (human, chimp, mouse, rat and dog) as well as in chicken; two repeats were found in intron 16 of the frog; and one repeat in the zebrafish. These data suggest that Fox-2 may be important for regulating exon 16 splicing from fish to man. Next, to test whether a similar mechanism might regulate other tissue-specific exons, we analyzed a larger group of some 27 alternative exons with predominant expression in the brain. Remarkably, 80–90% of these exons possessed one or more UGCAUG motifs within 1kb of flanking intron sequence. The majority of these elements were concentrated in the intron 10–400nt downstream of the regulated exons. Phylogenetic analysis of individual exons revealed that the number and position of intronic UGCAUG elements were highly conserved among mammalian species and in the chicken, but more divergent in fish. In marked contrast, control datasets of constitutively spliced exons, and alternatively spliced exons not known to be regulated in tissue-specific patterns, exhibited a low incidence of UGCAUG elements in the flanking introns. These findings are exactly as expected for a functionally important regulatory element. We propose that the high binding specificity of Fox proteins, and the unique localization of UGCAUG enhancers near regulated alternative exons, is advantageous for splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in specific cell types including erythroblasts. We speculate that erythroid-specific splicing is accomplished by coordination between Fox-2 and other as yet unknown splicing factors.

New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.

The increase in genome scanning data, derived from clinical genetics practice, is producing a wealth of information on human sequence variability. The critical issue is to identify if a given nucleotide change results in a benign polymorphism or a disease-causing mutation. We have focused on one specific gene expression step, pre-mRNA processing, where we can functionally define the effect of nucleotide changes and in turn the patient's mutation can shed light on the basic pre mRNA splicing mechanisms. Our results show that several nucleotide changes in CFTR exon 12 induce a variable extent of exon skipping that leads to reduced levels of normal transcripts. This is the case in both natural mutations D565G and G576A (the latter having previously considered a neutral polymorphism) and several site-directed silent substitutions. We demonstrate here that this phenomenon is due to the interference with a new regulatory element that we have named composite exonic regulatory element of splicing (CERES). The effect of single nucleotide substitutions at CERES cannot be predicted by neither SR matrices nor enhancer identification. The recognition and characterization of splicing abnormalities, caused by exon sequence variations at CERES elements, may represent a frequent disease-causing mechanism that also relates to the phenotypic variability. Our results indicate that even the most benign looking polymorphism in an exon cannot be ignored as it may affect the splicing process. Hence, appropriate functional splicing assays should be included in genotype screenings to distinguish between polymorphisms and pathogenic mutations.

Trypanosoma cruzi TcSRPK, the first protozoan member of the SRPK family, is biochemically and functionally conserved with metazoan SR protein-specific kinases

A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.