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.     

An intronic polypyrimidine-rich element downstream of the donor site modulates cystic fibrosis transmembrane conductance regulator exon 9 alternative splicing

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041-21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774-1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5'-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5'-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5'-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3'-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5'-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5'-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3'-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1.     

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(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.     

SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer

The intronic splicing silencer (ISS) of CFTR exon 9 promotes exclusion of this exon from the mature mRNA. This negative influence has important consequences with regards to human pathologic events, as lack of exon 9 correlates well with the occurrence of monosymptomatic and full forms of CF disease. We have previously shown that the ISS element interacts with members of the SR protein family. In this work, we now provide the identification of SF2/ASF and SRp40 as the specific SR proteins binding to this element and map their precise binding sites in IVS9. We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems. Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a ‘decoy’ sequence in the vicinity of its suboptimal donor site. The results of this study give an insight on intron ‘exonization’ mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.     

An exonic splicing silencer in the testes-specific DNA ligase III beta exon

Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation.     

Muscle-specific exonic splicing silencer for exon exclusion in human ATP synthase gamma-subunit pre-mRNA.

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.     

An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3

Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site.     

hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure

The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.     

Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12

In general, splicing regulatory elements are defined as Enhancers or Silencers depending on their positive or negative effect upon exon inclusion. Often, these sequences are usually present separate from each other in exonic/intronic sequences. The Composite Exonic Splicing Regulatory Elements (CERES) represent an extreme physical overlap of enhancer/silencer activity. As a result, when CERES elements are mutated the consequences on the splicing process are difficult to predict. Here, we show that the functional activity of the CERES2 sequence in CFTR exon 12 is regulated by the binding, in very close proximity to each other, of several SR and hnRNP proteins. Moreover, our results show that practically the entire exon 12 sequence context participate in its definition. The consequences of this situation can be observed at the evolutionary level by comparing changes in conservation of different splicing elements in different species. In conclusion, our study highlights how it is increasingly difficult to define many exonic sequences by simply breaking them down in isolated enhancer/silencer or even neutral elements. The real picture is close to one of continuous competition between positive and negative factors where affinity for the target sequences and other dynamic factors decide the inclusion or exclusion of the exon.     

CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF⁶⁵ and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain.     

Missense,nonsense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3' portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms.     

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.     

Simultaneous cycle sequencing assessment of (TG)m and Tn tract length in CFTR gene

The lengths of the dinucleotide (TG)m and mononucleotide Tn repeats, both located at the intron 8/exon 9 splice acceptor site of the cystic fibrosis transmembrane conductance regulator (CFTR) gene whose mutations cause cysticfibrosis (CF), have been shown to influence the skipping of exon 9 in CFTR mRNA. This exon 9-skipped mRNA encodes a nonfunctional protein and is associated with various clinical manifestations in CF As a result of growing interest in these repeats, several assessment methods have been developed, most of which are, however, cumbersome, multi-step, and time consuming. Here, we describe a rapid methodfor the simultaneous assessment of the lengths of both (TG)m and Tn repeats, based on a nonradioactive cycle sequencing procedure that can be performed even without DNA extraction. This method determines the lengths of the (TG)m and Tn tracts of both alleles, which in our samples ranged from TG8 to TG12 in the presence of T5, T7, and T9 alleles, and also fully assesses the aplotypes. In addition, the repeats in the majority of these samples can be assessed by single-strand sequencing, with no need to sequence the other strand, thereby saving a considerable amount of time and effort.     

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.     

TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.     

Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins.

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.