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Emanuele Buratti

Publications and source records attributed to Emanuele Buratti.

At least 19 recordsLinked to original sources
PubMed2025

Rescue of common and rare exon 2 skipping variants of the GAA gene using modified U1 snRNA.

BACKGROUND: Pompe disease (PD) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of acid alpha glucosidase (GAA) enzyme due to mutations in the GAA gene. As a result, undigested glycogen accumulates within lysosomes causing their dysfunction. From a clinical point of view, the disease can be classified in infantile-onset (IO) and late-onset (LO) forms. The common GAA c.-32-13T>G variant, found in 40-70% of LO-PD alleles, is a leaky splicing mutation interfering with the correct GAA exon 2 recognition by the spliceosome leading to the production of non-functional GAA transcripts. In this study, we used modified, GAA-tailored U1 snRNAs to correct the aberrant splicing determined by the c.-32-13T>G and other GAA exon 2-skipping mutations. METHODS: A set of constructs expressing 5 different engineered U1 snRNAs was generated. A functional splicing assay using a GAA hybrid minigene carrying different variants known to affect GAA exon 2 splicing was used to test the effect of engineered U1 snRNAs on exon 2 inclusion. The effect on endogenously expressed GAA transcript and GAA enzymatic activity was assessed by transfecting patient-derived fibroblasts bearing the common c.-32-13T>G with the best performing modified U1 snRNA. RESULTS: Modified U1-3, U1+1 and U1+6 snRNAs were all able to increase, in a dose-dependent manner, the inclusion of exon 2 within the transcript derived from the GAA minigene harbouring the c.-32-13T>G variant. The U1+1 was the most effective one (2,5 fold increase). Moreover, U1+1 snRNA partially rescued the correct splicing of GAA minigenes harbouring mutations that affect the 3'ss (c.-32-3C>G, c.-32-2A>G) and the 5'ss (c.546G>A, c.546G>C, c.546G>T). Notably, the treatment of patient-derived fibroblasts carrying the c.-32-13T>G mutation with the U1+1 snRNA increased the amount of normal GAA mRNA by 1,8 fold and the GAA enzymatic activity by 70%. CONCLUSIONS: we provide the proof-of-concept for the use of modified GAA-tailored U1 snRNAs, designed to potentiate the recognition of the GAA exon 2 5'ss, as therapeutic tools to correct the aberrant transcripts carrying variants that affect exon 2 splicing, including the common c.-32-13T>G variant.

Humans↗
PubMed2006

Defective splicing, disease and therapy: searching for master checkpoints in exon definition.

The number of aberrant splicing processes causing human disease is growing exponentially and many recent studies have uncovered some aspects of the unexpectedly complex network of interactions involved in these dysfunctions. As a consequence, our knowledge of the various cis- and trans-acting factors playing a role on both normal and aberrant splicing pathways has been enhanced greatly. However, the resulting information explosion has also uncovered the fact that many splicing systems are not easy to model. In fact we are still unable, with certainty, to predict the outcome of a given genomic variation. Nonetheless, in the midst of all this complexity some hard won lessons have been learned and in this survey we will focus on the importance of the wide sequence context when trying to understand why apparently similar mutations can give rise to different effects. The examples discussed in this summary will highlight the fine 'balance of power' that is often present between all the various regulatory elements that define exon boundaries. In the final part, we shall then discuss possible therapeutic targets and strategies to rescue genetic defects of complex splicing systems.

Exons↗
PubMed2006

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region.

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.

Alternative Splicing↗
PubMed2006

Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon.

In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

Base Sequence↗
PubMed2006

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.

Aged↗
PubMed2005

Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene.

Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)16 tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.

Apolipoprotein A-II↗
PubMed2005

TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing.

TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly.

Alternative Splicing↗
PubMed2005

Human, Drosophila, and C.elegans TDP43: nucleic acid binding properties and splicing regulatory function.

TAR DNA binding protein (TDP43), a highly conserved heterogeneous nuclear ribonucleoprotein, was found to down-regulate splicing of the exon 9 cystic fibrosis transmembrane conductance regulator (CFTR) through specific binding to a UG-rich polymorphic region upstream of the 3' splice site. Despite the emergence of new information regarding the protein's nuclear localization and splicing regulatory activity, TDP43's role in cells remains elusive. To investigate the function of human TDP43 and its homologues, we cloned and characterized the proteins from Drosophila melanogaster and Caenorhabditis elegans. The proteins from human, fly, and worm show striking similarities in their nucleic acid binding specificity. We found that residues at two different positions, which show a strong conservation among TDP43 family members, are linked to the tight recognition of the target sequence. Our three-dimensional model of TDP43 in complex with a (UG)(m) sequence predicts that these residues make amino acid side-chain to base contacts. Moreover, our results suggest that Drosophila TDP43 is comparable to human TDP43 in regulating exon splicing. On the other hand, C.elegans TDP43 has no effect on exon recognition. TDP43 from C.elegans lacks the glycine-rich domain found at the carboxy terminus of the other two homologues. Mutants of human and fly TDP43 devoid of the C-terminal domain are likewise unable to affect splicing. Our studies suggest that the glycine-rich domain is essential for splicing regulation by human and fly TDP43.

Amino Acid Sequence↗
PubMed2005

A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD).

The different alleles at the (TG)m(T)n polymorphic loci at the 3' end of the human CFTR intron 8 determine the efficiency by which exon 9 is spliced. We identified a novel TG12T3 allele in a congenital bilateral absence of vas deferens (CBAVD) patient who carries a [TG11T7; p.Phe508Cys; p.Met470Val] haplotype on the other chromosome. To better understand the complex regulation of exon 9 splicing, we analyzed the levels of correctly spliced CFTR transcripts in six CFTR-expressing epithelial cell lines derived from lung, colon, testis, vas deferens, and epididymis transiently transfected with four CFTR minigenes (pTG11T7, pTG12T7, pTG12T5, and pTG12T3). In this work, we show that a decrease in the Ts at the polymorphic locus in a TG12 background determines a cell-type dependent reduction in exon 9+ transcripts that is not related to the basal splicing efficiency in the cell line. These data emphasize the role of the T5 allele in CBAVD and identify the T3 allele as a severe cystic fibrosis (CF) disease-causing mutation. Finally, UV cross-linking experiments demonstrated that tissue-specific trans-acting splicing factors do not contribute to the different patterns of exon 9 splicing found between the cell lines. However, we observed that lower numbers of Ts can alter the binding of TDP-43 (TDP43 or TARDBP) to its specific target ug12 in a tissue-specific manner. Our results support the idea that the ratio of general splicing factors plays a role in the tissue variability of exon 9 alternative splicing.

Adult↗
PubMed2005

Another step forward for SELEXive splicing.

Splicing alterations are being increasingly reported to cause human diseases. However, predicting beforehand whether a given mutation might lead to aberrant splicing is often hampered by insufficient knowledge of which cis-acting sequences affect exon recognition. To better define these sequences, experimental methods have been developed that integrate pre-mRNA splicing with sequence selection assays. Recently, a novel in vivo selection method based on a partially randomized full-length exon has enabled the identification of new splicing-controlling elements in SMN exon 7. Skipping of this exon results in an aberrant protein and is involved in proximal spinal muscular atrophy (SMA). Hopefully, this approach will provide novel targets for nascent RNA molecular medicine approaches for the recovery of abnormal pre-mRNA splicing events.

Alternative Splicing↗
PubMed2004

hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes.

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA-protein complexes at several 5' splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5' splice site (5'ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5'ss of TSHbeta exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.

Binding Sites↗
PubMed2004

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.

Alternative Splicing↗
PubMed2004

Ribosomal protein S1 specifically binds to the 5' untranslated region of the Pseudomonas aeruginosa stationary-phase sigma factor rpoS mRNA in the logarithmic phase of growth.

The rpoS gene encodes the stationary-phase sigma factor (RpoS or sigma(s)), which was identified in several gram-negative bacteria as a central regulator controlling the expression of genes involved in cell survival in response to cessation of growth (stationary phase) and providing cross-protection against various stresses. In Pseudomonas aeruginosa, the levels of sigma(s) increase dramatically at the onset of the stationary phase and are regulated at the transcriptional and posttranscriptional levels. The P. aeruginosa rpoS gene is transcribed as a monocistronic rpoS mRNA transcript comprised of an unusually long 373-bp 5' untranslated region (5' UTR). In this study, the 5' UTR and total protein extracts from P. aeruginosa logarithmic and stationary phases of growth were used in order to investigate the protein-RNA interactions that may modulate the translational process. It was observed that a 69-kDa protein, which corresponded to ribosomal protein S1, preferentially binds the 5' UTR of the rpoS mRNA in the logarithmic phase and not in the stationary phase. This is the first report of a protein-rpoS mRNA 5' UTR interaction in P. aeruginosa, and the possible involvement of protein S1 in translation regulation of rpoS is discussed.

5' Untranslated Regions↗
PubMed2004

RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA exon.

In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.

Alternative Splicing↗
PubMed2003

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.

Alternative Splicing↗
PubMed2003

Can a 'patch' in a skipped exon make the pre-mRNA splicing machine run better?

It is becoming clear that exonic sequences can act as determinants of their own fate: the inclusion or exclusion from mature mRNA. Indeed, even silent nucleotide substitutions can cause aberrant exon skipping, resulting in a disease phenotype. It might be possible to restore essential splicing functions, lost through mutations, using molecular therapy at the RNA level. A variety of methods have been attempted, the most promising being the recent use of chimeric compounds that localize splicing-functional peptides by base complementarity.

Alternative Splicing↗