Franco Pagani

Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis‐acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3′ splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP‐43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP‐43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP‐43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(ΔF508)/TG13T3(wt) genotype leading to a disease‐causing high proportion of exon 9 skipping.

Genomic variants in exons and introns: identifying the splicing spoilers

When genome variants are identified in genomic DNA, especially during routine analysis of disease-associated genes, their functional implications might not be immediately evident. Distinguishing between a genomic variant that changes the phenotype and one that does not is a difficult task. An increasing amount of evidence indicates that genomic variants in both coding and non-coding sequences can have unexpected deleterious effects on the splicing of the gene transcript. So how can benign polymorphisms be distinguished from disease-associated splicing mutations?

A new type of mutation causes a splicing defect in ATM.

Disease-causing splicing mutations described in the literature primarily produce changes in splice sites and, to a lesser extent, variations in exon-regulatory sequences such as the enhancer elements. The gene ATM is mutated in individuals with ataxia-telangiectasia; we have indentified the aberrant inclusion of a cryptic exon of 65 bp in one affected individual with a deletion of four nucleotides (GTAA) in intron 20. The deletion is located 12 bp downstream and 53 bp upstream from the 5′ and 3′ ends of the cryptic exon, respectively. Through analysis of the splicing defect using a hybrid minigene system, we identified a new intron-splicing processing element (ISPE) complementary to U1 snRNA, the RNA component of the U1 small nuclear ribonucleoprotein (snRNP). This element mediates accurate intron processing and interacts specifically with U1 snRNP particles. The 4-nt deletion completely abolished this interaction, causing activation of the cryptic exon. On the basis of this analysis, we describe a new type of U1 snRNP binding site in an intron that is essential for accurate intron removal. Deletion of this sequence is directly involved in the splicing processing defect.

Regulation of TMEM16A chloride channel properties by alternative splicing

Expression of TMEM16A protein is associated with the activity of Ca2+-activated Cl− channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca2+ sensitivity by nearly 4-fold, resulting in Cl− currents requiring lower Ca2+ concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca2+-activated Cl− channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca2+ dependence of the TMEM16A-dependent Cl− channels in a tissue-specific manner.

Nuclear factor TDP-43 binds to the polymorphic TG repeats in CFTR intron 8 and causes skipping of exon 9: a functional link with disease penetrance.

To the Editor: In the January 2004 issue of AJHG, Groman et al. published a collaborative study on the disease penetrance of a common abbreviated tract of five thymidines (T5) in intron 8 of the CFTR gene (MIM 602421). By analyzing a large number of affected individuals presenting with male infertility or nonclassical cystic fibrosis (CF [MIM 219700]), they found that the pathogenic or benign effect of T5 correlated with variations in a TG repeat sequence just upstream of the polypyrymidines. In particular, longer TG repeats (12 or 13) are associated with disease phenotypes, and 91% of affected individuals, as opposed to 22% of unaffected ones, have repetitions of 12 or 13 TGs. Although the modifier effect of the TG repeats on the T5 variant has been proposed elsewhere (Cuppens et al. 1998), the principal novelty of the Groman et al. study lies in the high number of patients analyzed from different regions. As the T5 allele is found in ∼10% of the general population, the results of this study strongly indicate that determination of the TG repeats may have a significant diagnostic value. The pathologies studied by Groman et al. are in line with the growing number of human diseases resulting from pre-mRNA splicing alterations and, specifically, those diseases caused by mutations in cis-acting elements (the TG and T repeats) that disrupt the use of alternative splice sites, as recently reviewed by Faustino and Cooper (2003). In this specific case, the pathogenetic role of these repeats is owing to their effects on CFTR exon 9 at the level of pre-mRNA splicing, with longer UG repeats (and short U tracts) increasing exon 9 skipping and leading to the production of a nonfunctional protein (Delaney et al. 1993; Strong et al. 1993). An obvious question, not taken into consideration in the study by Groman et al., is what factor(s) or mechanism(s) determine the TG repeat number to be a reliable predictor of penetrance for the T5 allele. Indeed, some mechanisms have been proposed already, and, >2 years ago, we reported that TDP-43 binds specifically to the UG repeat sequence and, in this way, promotes skipping of CFTR exon 9 (Buratti et al. 2001). In the same study, overexpression of TDP-43 increased CFTR exon 9 skipping in a minigene system, and ablation of its expression by antisense oligonucleotides resulted in increased inclusion of the same exon. Moreover, TDP-43’s binding to the UG repeat sequence has been shown to correlate well with repeat length, and this has provided a clear rationale as to why longer UG tracts determine a higher rate of exon 9 skipping (Buratti and Baralle 2001). In this respect, it may be worthwhile also to point out that an independent and recent study confirmed that the mouse homologue of human TDP-43 (mTDP-43) also inhibits human CFTR exon 9 splicing in a minigene system (Wang et al. 2004). To demonstrate that the UG–TDP-43 interaction plays a direct role in splicing, we set up an in vitro splicing assay, a useful system to define the details of the molecular mechanisms involved and the role played by individual splicing factors. To this end, we introduced different TG repeats followed by 5T in the 3′ splice site in a heterologous context composed by two tropomyosin exons separated by a 111-nt intron sequence (see boxed sequences in fig. 1A). In vitro analysis of the splicing pattern of the transcribed RNA from these plasmids shows that UG elements repress splicing. In fact, increasing the number of UG repeats (UG6, UG8, and UG12) results in progressive splicing inhibition (fig. 1B). This direct role of UG repeats in repressing splicing also can be observed following the addition of an unlabeled (UG)12 RNA competitor to the splicing mix: as shown in figure 1C, this action can restore splicing activity of the UG12U5 RNA. As a control, this figure also shows that addition of cold (UG)12 RNA oligonucleotide does not have any effect on the splicing of the PY7(wt) construct (which does not contain any UG repeats) and that the addition of equal amounts of a control 30-mer RNA oligonucleotide has no effect on both the PY7(wt) and the PY7(UG12U5) RNAs. Figure 1 Interaction of UG repeats with TDP-43, causing splicing inhibition. A, Diagram of the in vitro splicing assay. Boxes indicate exons, and the horizontal line indicates intronic sequences. In the UG6U5 construct, an A residue was removed in an attempt to ... Having determined that (UG)12 sequences can promote splicing inhibition in vitro, we then checked whether depletion of TDP-43 from the splicing mix (fig. 1D) results in activation of splicing. Figure 1E shows that, indeed, depletion of TDP-43 from the nuclear extract results in activation of splicing in the UG12U5 RNA, an activation that can be abolished in the depleted extracts by the addition of recombinant TDP-43 (GST-TDP43). In conclusion, all these results are consistent with the model in which the TG repeats in the CFTR intron 8 bind to TDP-43, and this protein, in turn, inhibits splicing of exon 9. Taken together, our results provide not only a mechanistic explanation for the association data of Groman et al. but also may provide a basis to explain the variable phenotypic penetrance of the TG repeats. In fact, individual and tissue-specific variability in the concentration of this inhibitory splicing factor (Buratti et al. 2001) may explain the not-yet-complete penetrance of the TG tract in 22% of the unaffected individuals with the T5 variant and a high TG number (Groman et al. 2004) and may even represent one of the possible factors that determine whether an individual will develop multisystemic (nonclassical CF) or monosymptomatic (CBAVD) disease.

An IRF8-binding promoter variant and AIRE control CHRNA1 promiscuous expression in thymus.

Promiscuous expression of tissue-restricted auto-antigens in the thymus imposes T-cell tolerance and provides protection from autoimmune diseases. Promiscuous expression of a set of self-antigens occurs in medullary thymic epithelial cells and is partly controlled by the autoimmune regulator (AIRE), a nuclear protein for which loss-of-function mutations cause the type 1 autoimmune polyendocrine syndrome. However, additional factors must be involved in the regulation of this promiscuous expression. Here we describe a mechanism controlling thymic transcription of a prototypic tissue-restricted human auto-antigen gene, CHRNA1. This gene encodes the α-subunit of the muscle acetylcholine receptor, which is the main target of pathogenic auto-antibodies in autoimmune myasthenia gravis. On re-sequencing the CHRNA1 gene, we identified a functional bi-allelic variant in the promoter that is associated with early onset of disease in two independent human populations (France and United Kingdom). We show that this variant prevents binding of interferon regulatory factor 8 (IRF8) and abrogates CHRNA1 promoter activity in thymic epithelial cells in vitro. Notably, both the CHRNA1 promoter variant and AIRE modulate CHRNA1 messenger RNA levels in human medullary thymic epithelial cells ex vivo and also in a transactivation assay. These findings reveal a critical function of AIRE and the interferon signalling pathway in regulating quantitative expression of this auto-antigen in the thymus, suggesting that together they set the threshold for self-tolerance versus autoimmunity.

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre‐messenger RNA splicing

The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single‐nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre‐messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild‐type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO‐K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild‐type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild‐type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and single‐nucleotide polymorphisms at pre‐mRNA splicing and protein processing/functional levels. We show that aberrant pre‐mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation‐specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency. (HEPATOLOGY 2008.)

Regulation of Fibronectin EDA Exon Alternative Splicing: Possible Role of RNA Secondary Structure for Enhancer Display

ABSTRACT The fibronectin primary transcript undergoes alternative splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more complex IIICS region. We have shown previously that an 81-nucleotide region within the EDA exon is necessary for exon recognition and that this region contains at least two splicing-regulatory elements: a polypurinic enhancer (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showing that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-structure enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA conformation and raise the possibility that the display of the ESE element in a loop position may represent a significant feature of the exon splicing-regulatory region.

Promoter Architecture Modulates CFTR Exon 9 Skipping

Using hybrid minigene experiments, we have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, cystic fibrosis transmembrane regulator (CFTR) exon 9 and fibronectin extra domain-A (EDB). A specific alternative splicing pattern corresponded to each analyzed promoter. Promoter-dependent sensitivity to cotransfected regulatory splicing factor SF2/ASF was observed only for the CFTR exon 9, whereas that of the EDB was refractory to promoter-mediated regulation. Deletion in the CFTR minigene of the downstream intronic splicing silencer element binding SF2/ASF abolished the specific promoter-mediated response to this splicing factor. A systematic analysis of the regulatory cis-acting elements showed that in the presence of suboptimal splice sites or by deletion of exonic enhancer elements the promoter-dependent sensitivity to splicing factor-mediated inhibition was lost. However, the basal regulatory effect of each promoter was preserved. The complex relationships between the promoter-dependent sensitivity to SF2 modulated by the exon 9 definition suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation.

A Rare SMN2 Variant in a Previously Unrecognized Composite Splicing Regulatory Element Induces Exon 7 Inclusion and Reduces the Clinical Severity of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by homozygous inactivation of the SMN1 (Survival Motor Neuron 1) gene. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene from which only low amounts of full‐length mRNA are produced. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We identified and characterized the rare variant c.859G>C (p.Gly287Arg) in exon 7 in both SMN2 copies of a male patient affected with type III SMA, a milder form of the disease rarely associated with only two SMN2 copies. We demonstrated in vivo, in this patient and in a second unrelated patient, and ex vivo, using SMN splicing assays, that the variant induces inclusion of exon 7 into SMN2 mRNA. Moreover, we show that the c.859G>C variation is located in a composite splicing regulatory element in the centre of exon 7. The variation does not affect binding of HTra2â nor creates a novel SF2/ASF enhancer, but disrupts an hnRNP A1 binding site. The natural occurrence of enhanced inclusion of SMN2 exon 7 in milder SMA cases supports the current therapeutic strategies based on splicing modulation in SMA patients.