Unusual splice site mutations disrupt FANCA exon 8 definition

The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position − 3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition.     

Identification of common genetic variation that modulates alternative splicing

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.     

Associations between intronic non-B DNA structures and exon skipping

Non-B DNA structures are abundant in the genome and are often associated with critical biological processes, including gene regulation, chromosome rearrangement and genome stabilization. In particular, G-quadruplex (G4) may affect alternative splicing based on its ability to impede the activity of RNA polymerase II. However, the specific role of non-B DNA structures in splicing regulation still awaits investigation. Here, we provide a genome-wide and cross-species investigation of the associations between five non-B DNA structures and exon skipping. Our results indicate a statistically significant correlation of each examined non-B DNA structures with exon skipping in both human and mouse. We further show that the contributions of non-B DNA structures to exon skipping are influenced by the occurring region. These correlations and contributions are also significantly different in human and mouse. Finally, we detailed the effects of G4 by showing that occurring on the template strand and the length of G-run, which is highly related to the stability of a G4 structure, are significantly correlated with exon skipping activity. We thus show that, in addition to the well-known effects of RNA and protein structure, the relative positional arrangement of intronic non-B DNA structures may also impact exon skipping.     

Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1gamma gene

Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U)GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U)GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1γ gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.     

An intronic structure enabled by a long-distance interaction serves as a novel target for splicing correction in spinal muscular atrophy

Here, we report a long-distance interaction (LDI) as a critical regulator of alternative splicing of Survival Motor Neuron 2 (SMN2) exon 7, skipping of which is linked to spinal muscular atrophy (SMA), a leading genetic disease of children and infants. We show that this LDI is linked to a unique intra-intronic structure that we term internal stem through LDI-1 (ISTL1). We used site-specific mutations and Selective 2′-Hydroxyl Acylation analyzed by Primer Extension to confirm the formation and functional significance of ISTL1. We demonstrate that the inhibitory effect of ISTL1 is independent of hnRNP A1/A2B1 and PTB1 previously implicated in SMN2 exon 7 splicing. We show that an antisense oligonucleotide-mediated sequestration of the 3′ strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, a SMN-interacting protein, in SMA patient cells. Our results also reveal that the 3′ strand of ISTL1 and upstream sequences constitute an inhibitory region that we term intronic splicing silencer N2 (ISS-N2). This is the first report to demonstrate a critical role of a structure-associated LDI in splicing regulation of an essential gene linked to a genetic disease. Our findings expand the repertoire of potential targets for an antisense oligonucleotide-mediated therapy of SMA.     

An intronic signal for alternative splicing in the human genome

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3' splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.     

Haplotype-specific <Emphasis Type="Italic">MAPT</Emphasis> exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Background

Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.

Methods

In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.

Results

Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.

Conclusions

We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

     

TDP-43 and NOVA-1 RNA-binding proteins as competitive splicing regulators of the schizophrenia-associated TNIK gene

The RNA-binding protein TDP-43, associated to amyotrophic lateral sclerosis and frontotemporal dementia, regulates the alternative splicing of several genes, including the skipping of TNIK exon 15. TNIK, a genetic risk factor for schizophrenia and causative for intellectual disability, encodes for a Ser/Thr kinase regulating negatively F-actin dynamics.Here we show that in the human adult nervous system TNIK exon 15 is mostly included compared to the other tissues and that, during neuronal differentiation of human induced pluripotent stem cells and of human neuroblastoma cells, TNIK exon 15 inclusion increases independently of TDP-43 protein content. By studying the possible molecular interplay of TDP-43 with brain-specific splicing factors, we found that the neuronal NOVA-1 protein competitively inhibits both TDP-43 and hnRNPA2/B1 skipping activity on TNIK by means of a RNA-dependent interaction and that this competitive mechanism is common to other TDP-43 RNA targets. We also show that the TNIK protein isoforms including/excluding exon 15 differently regulate cell spreading in non-neuronal cells and neuritogenesis in primary cortical neurons.Our data suggest a complex regulation between the ubiquitous TDP-43 and the neuron-specific NOVA-1 splicing factors in the brain that may help better understand the pathobiology of both neurodegenerative diseases and schizophrenia.