Franco Pagani - Academia.edu (original) (raw)

Papers by Franco Pagani

Human Molecular Genetics, 1996

Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl est... more Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G 3′ splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (∆205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5′ splice site mutation (G to A) that leads to skipping of exon 8 (∆254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an XbaI restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and ∆254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.

Nucleic Acids Research

In disease-associated genes, the understanding of the functional significance of deep intronic nu... more In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5 0 and 3 0 splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a $40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5 0 -3 0 order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived fr... more Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the inclusion of the V region, and in particular the V25 alternative variant, was significantly higher in all fetal than in adult tis-

Journal of Biological Chemistry, 2003

Promoter-mediated regulation of alternative splicing 2 SUMMARY We have investigated the role of t... more Promoter-mediated regulation of alternative splicing 2 SUMMARY We have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, CFTR exon 9 and fibronectin EDB using hybrid minigene experiments. To each analyzed promoter corresponded a specific alternative splicing pattern. 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 (ISS) 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 element, 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 promoterdependent sensitivity to SF2\ASF modulated by the exon 9 definition, suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation. The abbreviations used are: CFTR, Cystic Fibrosis Transmembrane Regulator; FN EDB, Fibronectin Extra Domain-B; FN EDA, Fibronectin Extra Domain-A; ISS, intronic splicing silencer; ESS, exonic splicing silencer; ESE exonic splicing enhancer; pol II, polymerase II; CTD, C-terminal domain; SR proteins, Serine arginine-rich proteins; SF2/ASF, splicing factor 2.

American Journal of Human Genetics, 2004

... 1998 ), the principal novelty of the Groman et al. ... 1B). This direct role of UG repeats in... more ... 1998 ), the principal novelty of the Groman et al. ... 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 ...

Febs Letters, 2006

CFTR exon 9 presents a 3 0 splice site polymorphism, (UG) m U n , whose composition influences sp... more CFTR exon 9 presents a 3 0 splice site polymorphism, (UG) m U n , whose composition influences splicing. TDP43 specifically binds the UG tract of the transcript and inhibits splicing in vitro. We report that depletion of TDP43 through RNA interference removes splicing inhibition caused by unfavorable (UG)m U n sequences, indicating that TDP43 exerts a potent inhibitory effect in vivo. We also show that the UG-TDP43 interaction has a dominant role over other exon 9 splicing regulatory elements. These results suggest that TDP43 association near a splice site has determined the evolution of positive splicing regulatory elements to contrast this inhibition.

Embo Journal, 2001

Alternative splicing of human cystic ®brosis transmembrane conductance regulator (CFTR) exon 9 is... more Alternative splicing of human cystic ®brosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both¯anking introns (IVS8 and IVS9). Several studies have identi®ed 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 identi®ed that recognize this element. We have identi-®ed TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding speci®cally 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 ®nding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DF508)/ TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

Nucleic Acids Research, 2007

Alternative splicing has been associated with increased evolutionary changes and with recent exon... more Alternative splicing has been associated with increased evolutionary changes and with recent exon creation or loss. The addition of a new exon can be explained by its inclusion in only a fraction of the transcripts leaving the original form intact and giving to the new form the possibility to evolve independently but the exon loss phenomenon is less clear. To explore the mechanism that could be involved in CFTR exon 12 lower splicing efficiency in primates, we have analyzed the effect of multiple synonymous variations. Random patterns of synonymous variations were created in CFTR exon12 and the majority of them induced exon inclusion, suggesting a suboptimal splicing efficiency of the human gene. In addition, the effect of each single synonymous substitution on splicing is strongly dependent on the exonic context and does not correlate with available in silico exon splicing prediction programs. We propose that casual synonymous substitutions may lead to a reduced splicing efficiency that can result in a variable proportion of exon loss. If this phenomenon happens in in-frame exons and to an extent tolerated by the cells it can have an important evolutionary effect since it may generate a substrate for natural selection of new splicing isoforms.

Proceedings of The National Academy of Sciences, 2005

Abbreviation: CFTR, cystic fibrosis transmembrane regulator. *F.P. and M.R. contributed equally t... more Abbreviation: CFTR, cystic fibrosis transmembrane regulator. *F.P. and M.R. contributed equally to this work.

Febs Letters, 1996

Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression ... more Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglyeosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to Nglycosylation. A missense substitution, L273S, previously detected in a patient with eholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS ~ NML) indicated that the L273S mutation creates an additional N-glycosylation consensus (N-X-S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglyeosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N-glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri-oleyl glycerol and eholesteryl oleate by 50%, strongly suggesting that N-linked carbohydrate residues are important for optimal catalytic activity.

Journal of Biological Chemistry, 2000

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens,... more In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG m and T n polymorphic repeats at the 3 end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG m T n polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG m and T n polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG m and T n polymorphism at the 3 splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5 intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG m T n locus in monosymptomatic forms of cystic fibrosis.

Human Molecular Genetics, 2003

The increase in genome scanning data, derived from clinical genetics practice, is producing a wea... more 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.

Journal of Biological Chemistry, 2003

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupt... more 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.

Nature Reviews Genetics, 2004

European Journal of Epidemiology, 1992

The distribution of a polymorphism due to an Adenine to Guanine transition in the ApoAI gene has ... more The distribution of a polymorphism due to an Adenine to Guanine transition in the ApoAI gene has been studied in 136 women and 108 men, through amplification of the promoter region of the gene and allele-specific oligonucleotide hybridization. The allelic frequencies for the A allele were 0.10, 0.14 and 0.27 in women and 0.08, 017 and 0.14 in men for the lowest decile, intermediate group and the highest decile of HDL-cholesterol levels, respectively. Statistical analysis showed that the A allele was associated with high HDL-cholesterol levels in women, but not in men.

Nature, 2007

P LQ indicates patients with early onset of myasthenia gravis, defined by age at the onset of dis... more P LQ indicates patients with early onset of myasthenia gravis, defined by age at the onset of disease in the lower quartile of the distribution in each cohort (#21 yr in French patients, including 8 patients of age 21, and #18 yr in UK patients, including 10 patients of age 18). P UTQ indicates patients in the upper three quartiles of the distribution. C indicates matched controls. Odds ratios were determined relative to the control populations. The exact P-values were tested two-sided in the French cohort, and one-sided in the replication UK cohort. The combined data set was tested twosided using stratified 2 3 2 tables. The data also fitted a partially (or semi-) dominant model (Cochran-Armitage test with strata: P 5 4 3 10 24 ; odds ratio for AG versus AA genotypes, 2.27, 95% CI, 1.4-3.69; for GG versus AA genotypes, 5.15, 95% CI, 1.96-13.6).

Trends in Molecular Medicine, 2003

It is becoming clear that exonic sequences can act as determinants of their own fate: the inclusi... more 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.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign ... more Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G ¨ A in position Ϫ 1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the ⌬ 254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G ¨ A ( Ϫ 1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G ¨ A substitution observed in WD at position ϩ 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive ⌬ 254-277 protein.

Nature Genetics, 2002

The splicing processing defect in ATM intron 20 does not affect canonical splicing regulatory ele... more The splicing processing defect in ATM intron 20 does not affect canonical splicing regulatory elements. a, Genomic localization and sequence analysis of the ATM cryptic exon. Gray boxes, normal ATM exons; thin line, normal ATM introns; black box, position of the indicated nucleotide sequence of the ATM intron. The deletion of 4 bp (GTAA) in intron 20 (boxed) causes the activation of the cryptic 65-bp exon (uppercase). The splice sites used by the cryptic exon are underlined (the 5′ splice site did not have the almost universal GU donor site, but its weak GC variant). Inclusion of the cryptic exon out of the reading frame is predicted to lead to the insertion of 23 amino-acid residues followed by the introduction of a stop codon. Because of the peculiar nucleotide arrangement, the same final sequence can be obtained if the deletion is shifted in the 3′ direction by one base (TAAG) or two bases (AAGT). We did not find the deletion in 50 normal chromosomes. b, RT-PCR products obtained from total RNA and spanning exons 20-21 showed an aberrant splicing event (ins 65) in a lymphoblastoid cell line derived from the affected individual (lane 1) that is absent in a lymphoblastoid cell line derived from a control (lane 2). M, size marker (1-kb ladder). Radioactive semiquantitative RT-PCR analysis showed that about 20-30% of the total mRNA obtained was the aberrantly spliced product (data not shown). We did not see aberrant exon inclusion in ATM mRNA from normal individuals in heart, liver, lung colon, spleen kidney and skin fibroblasts. c, Allele-specific analysis of the RT-PCR product amplified with primers designed to amplify sequence spanning exons 16 (ex 16 dir) and 22 (ex 22 rev) and separated on an agarose gel. As the other mutant alleles encode the 2250G→A splicing mutation 25 , which results in complete in-frame skipping of exon 16, the experiment indicates that the reduced abundance of misspliced product could not be attributed to leaky splicing. d, Lower abundance of the aberrant splicing allele results from nonsense-mediated decay. Puromycin treatment increases the relative amount of the product containing the 65-nt insertion from cell line of the affected individual. RT-PCR products were amplified with primers designed to form products spanning exons 19-22 (ex 19 dir-ex 22 rev) and separated on an agarose gel. Lane 1, no puromycin; lane 2, pre-incubation with puromycin. e, The 4-bp deletion causes aberrant inclusion of the cryptic exon. Left, hybrid minigenes containing the indicated ATM variants. pATM-∆ contains the patient's genomic sequences with the 4-nt deletion. The two adenines of the deletion in the pATM-CT minigene were replaced with the CT dinucleotide. Intronic ATM sequences (heavy lines) are inserted in a hybrid minigene system 11,26 . Black and shaded boxes, α-globin and fibronectin sequences, respectively. Transcription is driven by the minimal α-globin promoter and SV40 enhancer (small arrows at 5′ end). The primers used in the RT-PCR assay are represented by arrows. The splicing products corresponding to either complete removal of the intron or inclusion of the cryptic exon are indicated.

Human Molecular Genetics, 1999

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) ... more The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3′ ′ ′ ′ splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG) n repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3′ ′ ′ ′ splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3′ ′ ′ ′ splice site sequences, the extent of exon 9 skipping in CFTR mRNA.

Human Molecular Genetics, 1996

Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl est... more Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G 3′ splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (∆205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5′ splice site mutation (G to A) that leads to skipping of exon 8 (∆254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an XbaI restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and ∆254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.

Nucleic Acids Research

In disease-associated genes, the understanding of the functional significance of deep intronic nu... more In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5 0 and 3 0 splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a $40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5 0 -3 0 order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived fr... more Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the inclusion of the V region, and in particular the V25 alternative variant, was significantly higher in all fetal than in adult tis-

Journal of Biological Chemistry, 2003

Promoter-mediated regulation of alternative splicing 2 SUMMARY We have investigated the role of t... more Promoter-mediated regulation of alternative splicing 2 SUMMARY We have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, CFTR exon 9 and fibronectin EDB using hybrid minigene experiments. To each analyzed promoter corresponded a specific alternative splicing pattern. 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 (ISS) 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 element, 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 promoterdependent sensitivity to SF2\ASF modulated by the exon 9 definition, suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation. The abbreviations used are: CFTR, Cystic Fibrosis Transmembrane Regulator; FN EDB, Fibronectin Extra Domain-B; FN EDA, Fibronectin Extra Domain-A; ISS, intronic splicing silencer; ESS, exonic splicing silencer; ESE exonic splicing enhancer; pol II, polymerase II; CTD, C-terminal domain; SR proteins, Serine arginine-rich proteins; SF2/ASF, splicing factor 2.

American Journal of Human Genetics, 2004

... 1998 ), the principal novelty of the Groman et al. ... 1B). This direct role of UG repeats in... more ... 1998 ), the principal novelty of the Groman et al. ... 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 ...

Febs Letters, 2006

CFTR exon 9 presents a 3 0 splice site polymorphism, (UG) m U n , whose composition influences sp... more CFTR exon 9 presents a 3 0 splice site polymorphism, (UG) m U n , whose composition influences splicing. TDP43 specifically binds the UG tract of the transcript and inhibits splicing in vitro. We report that depletion of TDP43 through RNA interference removes splicing inhibition caused by unfavorable (UG)m U n sequences, indicating that TDP43 exerts a potent inhibitory effect in vivo. We also show that the UG-TDP43 interaction has a dominant role over other exon 9 splicing regulatory elements. These results suggest that TDP43 association near a splice site has determined the evolution of positive splicing regulatory elements to contrast this inhibition.

Embo Journal, 2001

Alternative splicing of human cystic ®brosis transmembrane conductance regulator (CFTR) exon 9 is... more Alternative splicing of human cystic ®brosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both¯anking introns (IVS8 and IVS9). Several studies have identi®ed 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 identi®ed that recognize this element. We have identi-®ed TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding speci®cally 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 ®nding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DF508)/ TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

Nucleic Acids Research, 2007

Alternative splicing has been associated with increased evolutionary changes and with recent exon... more Alternative splicing has been associated with increased evolutionary changes and with recent exon creation or loss. The addition of a new exon can be explained by its inclusion in only a fraction of the transcripts leaving the original form intact and giving to the new form the possibility to evolve independently but the exon loss phenomenon is less clear. To explore the mechanism that could be involved in CFTR exon 12 lower splicing efficiency in primates, we have analyzed the effect of multiple synonymous variations. Random patterns of synonymous variations were created in CFTR exon12 and the majority of them induced exon inclusion, suggesting a suboptimal splicing efficiency of the human gene. In addition, the effect of each single synonymous substitution on splicing is strongly dependent on the exonic context and does not correlate with available in silico exon splicing prediction programs. We propose that casual synonymous substitutions may lead to a reduced splicing efficiency that can result in a variable proportion of exon loss. If this phenomenon happens in in-frame exons and to an extent tolerated by the cells it can have an important evolutionary effect since it may generate a substrate for natural selection of new splicing isoforms.

Proceedings of The National Academy of Sciences, 2005

Abbreviation: CFTR, cystic fibrosis transmembrane regulator. *F.P. and M.R. contributed equally t... more Abbreviation: CFTR, cystic fibrosis transmembrane regulator. *F.P. and M.R. contributed equally to this work.

Febs Letters, 1996

Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression ... more Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglyeosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to Nglycosylation. A missense substitution, L273S, previously detected in a patient with eholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS ~ NML) indicated that the L273S mutation creates an additional N-glycosylation consensus (N-X-S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglyeosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N-glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri-oleyl glycerol and eholesteryl oleate by 50%, strongly suggesting that N-linked carbohydrate residues are important for optimal catalytic activity.

Journal of Biological Chemistry, 2000

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens,... more In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG m and T n polymorphic repeats at the 3 end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG m T n polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG m and T n polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG m and T n polymorphism at the 3 splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5 intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG m T n locus in monosymptomatic forms of cystic fibrosis.

Human Molecular Genetics, 2003

The increase in genome scanning data, derived from clinical genetics practice, is producing a wea... more 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.

Journal of Biological Chemistry, 2003

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupt... more 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.

Nature Reviews Genetics, 2004

European Journal of Epidemiology, 1992

The distribution of a polymorphism due to an Adenine to Guanine transition in the ApoAI gene has ... more The distribution of a polymorphism due to an Adenine to Guanine transition in the ApoAI gene has been studied in 136 women and 108 men, through amplification of the promoter region of the gene and allele-specific oligonucleotide hybridization. The allelic frequencies for the A allele were 0.10, 0.14 and 0.27 in women and 0.08, 017 and 0.14 in men for the lowest decile, intermediate group and the highest decile of HDL-cholesterol levels, respectively. Statistical analysis showed that the A allele was associated with high HDL-cholesterol levels in women, but not in men.

Nature, 2007

P LQ indicates patients with early onset of myasthenia gravis, defined by age at the onset of dis... more P LQ indicates patients with early onset of myasthenia gravis, defined by age at the onset of disease in the lower quartile of the distribution in each cohort (#21 yr in French patients, including 8 patients of age 21, and #18 yr in UK patients, including 10 patients of age 18). P UTQ indicates patients in the upper three quartiles of the distribution. C indicates matched controls. Odds ratios were determined relative to the control populations. The exact P-values were tested two-sided in the French cohort, and one-sided in the replication UK cohort. The combined data set was tested twosided using stratified 2 3 2 tables. The data also fitted a partially (or semi-) dominant model (Cochran-Armitage test with strata: P 5 4 3 10 24 ; odds ratio for AG versus AA genotypes, 2.27, 95% CI, 1.4-3.69; for GG versus AA genotypes, 5.15, 95% CI, 1.96-13.6).

Trends in Molecular Medicine, 2003

It is becoming clear that exonic sequences can act as determinants of their own fate: the inclusi... more 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.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign ... more Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G ¨ A in position Ϫ 1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the ⌬ 254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G ¨ A ( Ϫ 1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G ¨ A substitution observed in WD at position ϩ 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive ⌬ 254-277 protein.

Nature Genetics, 2002

The splicing processing defect in ATM intron 20 does not affect canonical splicing regulatory ele... more The splicing processing defect in ATM intron 20 does not affect canonical splicing regulatory elements. a, Genomic localization and sequence analysis of the ATM cryptic exon. Gray boxes, normal ATM exons; thin line, normal ATM introns; black box, position of the indicated nucleotide sequence of the ATM intron. The deletion of 4 bp (GTAA) in intron 20 (boxed) causes the activation of the cryptic 65-bp exon (uppercase). The splice sites used by the cryptic exon are underlined (the 5′ splice site did not have the almost universal GU donor site, but its weak GC variant). Inclusion of the cryptic exon out of the reading frame is predicted to lead to the insertion of 23 amino-acid residues followed by the introduction of a stop codon. Because of the peculiar nucleotide arrangement, the same final sequence can be obtained if the deletion is shifted in the 3′ direction by one base (TAAG) or two bases (AAGT). We did not find the deletion in 50 normal chromosomes. b, RT-PCR products obtained from total RNA and spanning exons 20-21 showed an aberrant splicing event (ins 65) in a lymphoblastoid cell line derived from the affected individual (lane 1) that is absent in a lymphoblastoid cell line derived from a control (lane 2). M, size marker (1-kb ladder). Radioactive semiquantitative RT-PCR analysis showed that about 20-30% of the total mRNA obtained was the aberrantly spliced product (data not shown). We did not see aberrant exon inclusion in ATM mRNA from normal individuals in heart, liver, lung colon, spleen kidney and skin fibroblasts. c, Allele-specific analysis of the RT-PCR product amplified with primers designed to amplify sequence spanning exons 16 (ex 16 dir) and 22 (ex 22 rev) and separated on an agarose gel. As the other mutant alleles encode the 2250G→A splicing mutation 25 , which results in complete in-frame skipping of exon 16, the experiment indicates that the reduced abundance of misspliced product could not be attributed to leaky splicing. d, Lower abundance of the aberrant splicing allele results from nonsense-mediated decay. Puromycin treatment increases the relative amount of the product containing the 65-nt insertion from cell line of the affected individual. RT-PCR products were amplified with primers designed to form products spanning exons 19-22 (ex 19 dir-ex 22 rev) and separated on an agarose gel. Lane 1, no puromycin; lane 2, pre-incubation with puromycin. e, The 4-bp deletion causes aberrant inclusion of the cryptic exon. Left, hybrid minigenes containing the indicated ATM variants. pATM-∆ contains the patient's genomic sequences with the 4-nt deletion. The two adenines of the deletion in the pATM-CT minigene were replaced with the CT dinucleotide. Intronic ATM sequences (heavy lines) are inserted in a hybrid minigene system 11,26 . Black and shaded boxes, α-globin and fibronectin sequences, respectively. Transcription is driven by the minimal α-globin promoter and SV40 enhancer (small arrows at 5′ end). The primers used in the RT-PCR assay are represented by arrows. The splicing products corresponding to either complete removal of the intron or inclusion of the cryptic exon are indicated.

Human Molecular Genetics, 1999

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) ... more The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3′ ′ ′ ′ splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG) n repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3′ ′ ′ ′ splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3′ ′ ′ ′ splice site sequences, the extent of exon 9 skipping in CFTR mRNA.