Faouzi Baklouti

Faouzi Baklouti
Université Claude Bernard Lyon 1, Faculté de Médecine et de Pharmacie Lyon Est Institut NeuroMyoGène (INMG) Pathology and Genetics of Neuron and Muscle (PGNM) INSERM U1315, CNRS UMR 5261

Ph.D.

About

89
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Introduction
>Main interest: mRNA metabolism in normal and pathological cells >Previous and current projects: • mRNA metabolism in red blood cell membrane inherited disorders • Pre-mRNA alternative splicing regulation during eryhtroid differentiation • mRNA metabolism in malignant myelopathies • mRNA metabolism in neuromuscular pathologies

Publications

Publications (89)
Article
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Exon 16 inclusion is a critical splicing event that triggers the production of a functional protein 4.1R in mature normal erythroblasts, and is obviated in PU.1-induced erythroleukemia cells. Exon 16 contains an exonic splicing silencer (ESS16) that interacts with hnRNP A/B in heterologous cell context. We here show that ESS16 promotes the recruitm...
Article
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Blood Cancer Journal is a peer-reviewed, open access online journal publishing pre-clinical and clinical work in the field of hematology with ramifications into translational biology research down to new therapies
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In the January 2013 issue of this Journal, Joly and Colleagues have described an unusual case of sickle cell trait.1 The patient, originating from Ivory Coast, exhibits a low HbS level (12%) with no phenotypic manifestations. The mutation was found on a β-globin cluster haplotype of the Benin type. The Authors argued that the low percentage of HbS...
Article
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SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decre...
Data
SRSF5 knockdown and impact on pre-mRNA splicing in pre-differentiated MEL cells. EGFP-SRSF5 cells were transfected with siRNA specifically targeting SRSF5 transcripts. SRSF5 mRNAs and proteins were analyzed to assess the knockdown efficiency 24 and 48 h after transfection. Mock cells were transfected with irrelevant siRNA.A. Real-time RT-PCR. SRSF5...
Data
Primers used in this study. Mismatches (underlined sequences) were introduced to disrupt the ESE within exon 16 (F1 and R1), a stop codon in EGFP-SRSF5-ΔRS construct (R3), or to mutate Ser86 residue (S86A-S and S86A-AS). Heterologous sequences were added in 5′ of some primers (bolded), to create restriction sites (italic) for cloning purposes. F: f...
Data
Decreased expression of SRSF5 during erythroid differentiation. MEL cells were stably transfected with EGFP-SRSF5 construct and cultured in the absence (−) or presence (+) of DMSO for 4 days. Immunoblot analysis using mAB104 antibody reveals a dramatic and concomitant decrease of both endogenous SRSF5 and exogenous EGFP-SRSF5 in treated cells. (TIF...
Article
Complete loss of protein 4.1R in red blood cell membrane is a very rare condition in humans. We here explore the third case. The morphological and biochemical observations suggested that the proband suffers from homozygous hereditary elliptocytosis. Both parents, who are consanguineous, have an elliptocytosis with no cell fragmentation, typical of...
Article
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The -158 (C→T) nucleotide change, known as Xmn I polymorphism, occurs in (G)γ-globin gene promoter, and results in elevated fetal hemoglobin (HbF). We found this mutation in cis of a β(0)-thalassemia splicing mutation. Despite the complete absence of adult HbA, the phenotype was only moderately severe with no detectable alteration of α-globin gene...
Article
We describe a new approach to stabilize nonsense mRNA, based on the inhibition of the NMD mechanism, by combining cycloheximide-mediated inhibition of translation, and caffeine-mediated inhibition of UPF1 phosphorylation. This approach aimed to identify the impact of a 4.1R splicing mutation. This mutation is involved in a partial deficiency of 4.1...
Article
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Recessive mutations of the myosin VIIA (MYO7A) gene are reported to be responsible for both a deaf-blindness syndrome (Usher type 1B [USH1B] and atypical Usher syndrome) and nonsyndromic hearing loss (HL; Deafness, Neurosensory, Autosomal Recessive 2 [DFNB2]). The existence of DFNB2 is controversial, and often there is no relationship between the t...
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Spi-1/PU.1 oncogene is downregulated as proerythroblasts undergo terminal differentiation. Insertion of the Friend virus upstream of the Spi-1/PU.1 locus leads to the constitutive upregulation of Spi-1/PU.1, and a subsequent block in the differentiation of the affected erythroblasts. We have shown that sustained overexpression of Spi-1/PU.1 also in...
Article
We report on a Moroccan family in which the proposita displays a picture of β-thalassaemia intermedia, associated with heterozygous Hb O-Arab (β121 Glu → Lys) and a β°-thalassaemia trait. Hb O-Arab was ascertained by the disappearance of the Eco RI restriction site that, normally overlaps the β-globin gene codon 121. The proposita further presents...
Article
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Sustained expression of the Spi-1/PU.1 and Fli-1 oncoproteins blocks globin gene activation in mouse erythroleukemia cells; however, only Spi-1/PU.1 expression inhibits the inclusion of exon 16 in the mature 4.1R mRNA. This splicing event is crucial for a functional 4.1R protein and, therefore, for red blood cell membrane integrity. This report dem...
Article
It has long been considered that cryptic splice sites are ignored by the splicing machinery in the context of intact genuine splice sites. In the present study, it is shown that cryptic splice sites are utilized in all circumstances, when the authentic site is intact, partially functional or completely abolished. Their use would therefore contribut...
Article
We studied a large Swiss family with dominantly inherited hereditary spherocytosis and band 3 (anion exchanger 1, AE1) deficiency. Band 3 cDNA was analysed by single-strand conformation polymorphism analysis and nucleotide sequencing. A new point mutation was found: G771D (GGC→GAC). This change was present in all eight investigated patients but abs...
Article
An increasing number of genomic variations are no more regarded as harmless changes in protein coding sequences or as genetic polymorphisms. Studying the impact of these variations on mRNA metabolism became a central issue to better understand the biological significance of disease. We describe here a severe congenital muscular dystrophy (CMD) with...
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Congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of neuromuscular disorders, with autosomal recessive inheritance. We report a patient with severe congenital muscular dystrophy and total deficiency in the laminin alpha2 chain. Genetic analyses showed a linkage to the MDC1A locus for the patient's family, a...
Chapter
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During evolution, eukaryotic cells have developed an economic mechanism called pre-mRNA alternative splicing, to considerably increase the number of cellular functions, from a limited number of genomic loci. Recent insights into the pivotal role of RNA polymerase II in coupling transcription and virtually all the other steps of mRNA metabolism, hav...
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4.1R pre-mRNA alternative splicing results in multiple mRNA and protein isoforms that are expressed in virtually all tissues. More specifically, isoforms containing the alternative exon 17a, are exclusively expressed in muscle tissues. In this report, we show that these isoforms are preferentially present in the myoplasm of fast myofibres. 4.1R epi...
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Oncogene activity ranges from transduction signals to transcription factors. Altered expression of oncogenes, either by chromosomal translocation, proviral insertion or point mutations, can lead to tumor formation. More specifically, data accumulated through the last two decades have shown that disregulation of oncogenic transcription factors can i...
Article
Messenger RNAs containing premature stop codons are generally targeted for degradation through the nonsense-mediated mRNA decay (NMD) pathway. The subcellular localization of the NMD process in higher eukaryotes remains controversial. While many mRNAs are subjected to NMD prior to their release from the nucleus, a few display cytoplasmic NMD. To un...
Article
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The inclusion of exon 16 in mature protein 4.1R mRNA arises from a stage-specific splicing event that occurs during late erythroid development. We have shown that mouse erythroleukemia (MEL) cells reproduce this erythroid-specific splicing event upon induction of differentiation. We here found that this splicing event is regulated specifically in e...
Article
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The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20...
Article
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The inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleu...
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Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH express...
Article
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Early biochemical studies defined 4 functional domains of the erythroid protein 4.1 (4.1R). From amino-terminal to carboxy-terminal, these are 30 kd, 16 kd, 10 kd, and 22/24 kd in size. Although the functional properties of both the 30-kd and the 10-kd domain have been demonstrated in red cells, no functional activities have been assigned to either...
Article
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Protein 4.1 pre-mRNA splicing is regulated in tissue- and development-specific manners. Exon 16, which encodes the N-terminal region of the spectrin/actin-binding domain, is one of the alternatively spliced sequence motifs. It is present in late differentiated erythroid cells but absent from early erythroblasts and from lymphoid cells. We describe...
Article
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Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed...
Article
Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed...
Article
Protein 4.1 is a major component of the junctional complex at the red cell skeleton. Genomic studies have recently evidenced that the encoding gene (EL1 locus) is present in a single copy per haploid genome. Several RFLPs have already been characterized within intron sequences. Here, we describe the first RFLP found within the coding sequence. This...
Article
Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By ti...
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We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA-->TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele L...
Article
We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA-->TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele L...
Article
Full-text available
Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generate...
Article
Red cells ow their mechanical properties, that is, their resistance and their elastic deformability, to a protein network that laminates the lipid bilayer and to proteins spanning the latter. All proteins are interconnected. Their structure, as well as the structure of the corresponding genes, will be outlined. Numerous mutations have allowed to re...
Article
Investigations throughout the last decade have established that cytoskeleton integrity ensures red cell deformability and mechanical stability, and that defects in one of the skeletal components usually result in more or less severe hemolytic anemias. Although a large number of molecular defects have been identified to date, many others still bypas...
Article
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Protein 4.1 has been defined as a major component of the subcortical skeleton of erythrocytes. It binds the spectrin--actin scaffold through a 10-kD internal domain. This binding requires an essential 21-amino acid sequence motif, Motif I, which is retained by alternative splicing at the late stage of erythroid differentiation. We here analyze the...
Article
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The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Un...
Article
Heterozygous 4.1(-) hereditary elliptocytosis results from the absence of one haploid set of protein 4.1, a major component of the red cell skeleton. Two successive epidemiological investigations revealed fifteen probands in the French Northern Alps. The frequency of this disease seems to be very high in four small villages isolated in the Aravis m...
Article
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An asymptomatic shortened variant of protein 4.1 (-8.5 Kd) was first recognized in the red blood cells and designated protein 4.1 Presles. We show here that the missing segment belongs to the 22/24 Kd domain. Protein 4.1 cDNA from reticulocytes was amplified, mapped, and sequenced. The truncation appeared to result from the prevalent skipping of an...
Article
Full-text available
An asymptomatic shortened variant of protein 4.1 (-8.5 Kd) was first recognized in the red blood cells and designated protein 4.1 Presles. We show here that the missing segment belongs to the 22/24 Kd domain. Protein 4.1 cDNA from reticulocytes was amplified, mapped, and sequenced. The truncation appeared to result from the prevalent skipping of an...
Article
Full-text available
Spectrin Jendouba (alpha II/31) was found in a Tunisian family. In the heterozygous state, it is associated with asymptomatic elliptocytosis and a minimal defect in spectrin dimer self-association. On partial digestion of spectrin with trypsin, an abnormal cleavage appeared following Lys 788. Peptide and DNA sequencing indicated that the responsibl...
Article
Spectrin Jendouba (alpha II/31) was found in a Tunisian family. In the heterozygous state, it is associated with asymptomatic elliptocytosis and a minimal defect in spectrin dimer self-association. On partial digestion of spectrin with trypsin, an abnormal cleavage appeared following Lys 788. Peptide and DNA sequencing indicated that the responsibl...
Article
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Elliptocytogenic alpha I/36 spectrin Sfax is a new variant found in a Tunisian family. The alpha I/36 allele yielded a clinically manifest picture only when occurring in trans to a recently identified, low expression level polymorphism referred to as the alpha V/41 allele. Spectrin dimers were slightly increased in 4 degrees C extracts. On peptide...
Article
Elliptocytogenic alpha I/36 spectrin Sfax is a new variant found in a Tunisian family. The alpha I/36 allele yielded a clinically manifest picture only when occurring in trans to a recently identified, low expression level polymorphism referred to as the alpha V/41 allele. Spectrin dimers were slightly increased in 4 degrees C extracts. On peptide...
Article
Full-text available
4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE....
Article
Full-text available
4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE....
Article
Full-text available
Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existenc...
Article
A category of spectrin alpha I domain variants are manifested by the increase of the alpha I 74 kDa fragment at the expense of the parent 80 kDa fragment following partial tryptic digestion. We describe a particular case of alpha I/74 abnormality in a Tunisian family. The propositus was severely ill and had an elliptopoikilocytosis. To the contrary...
Article
The genetic disorders of the red cell skeleton encompass hereditary spherocytosis, hereditary elliptocytosis and an array of ill-defined haemolytic anaemias. Protein chemistry and molecular genetics have illuminated the supramolecular arrangement of the skeleton, the sequence and three-dimensional structure of its protein components, the exon-intro...
Article
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We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relat...
Article
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An atypical sickle cell trait with a very low level of hemoglobin S and features of heterozygous beta-thalassemia was recently described. In vitro globin chain synthesis strongly suggested the presence of the two abnormalities on the same chromosome. We report the corresponding beta S-thal gene. DNA sequence revealed a C----T base substitution in t...
Article
An atypical sickle cell trait with a very low level of hemoglobin S and features of heterozygous beta-thalassemia was recently described. In vitro globin chain synthesis strongly suggested the presence of the two abnormalities on the same chromosome. We report the corresponding beta S-thal gene. DNA sequence revealed a C----T base substitution in t...
Article
We have previously described the first homozygous cases of Hb Knossos in an Algerian family. The Hb A2 was completely absent, ascertaining the presence of a delta zero-thalassemia determinant in cis of the beta Knossos S gene. Here, we investigate the affected delta-globin gene. The complete DNA sequence of the gene and its 5' and 3' flanking regio...
Article
We have previously described the first homozygous cases of Hb Knossos in an Algerian family. The Hb A2 was completely absent, ascertaining the presence of a δ°-thalassemia determinant in cis of the βKnossoss gene. Here, we investigate the affected δ-globin gene. The complete DNA sequence of the gene and its 5′ and 3′ flanking regions was determined...
Article
A man in heart failure with a high oxygen affinity haemoglobin variant (Hb Rainier) underwent a mitral commissurotomy with the aid of cardiopulmonary bypass. Pre-operatively, a total blood exchange transfusion was carried out to prevent potential hypoxic and thrombo-embolic complications. No complications occurred in the postoperative period.
Article
Increased homotropic allosteric effect, while maintaining normal heterotropic effects, was observed in hemoglobin Loire. The oxygen binding curves, at equilibrium, and the kinetic measurements demonstrated that the substitution of alpha 88(F9) Ala for a Ser results in increased oxygen affinity and decreased n50 value. The function of the residues i...
Article
We searched an interaction between (i) pentoxifylline and/or propentofylline, and (ii) the red cell membrane with special emphasis on the membrane skeleton. It appeared (i) that propentofylline has no permanent binding site on the membrane, (ii) that propentofylline and/or pentoxifylline do not detectably alter spectrin conformation (no change of s...
Article
Increased homotropic allosteric effect, while maintaining normal heterotropic effects, was observed in hemoglobin Loire. The oxygen binding curves, at equilibrium, and the kinetic measurements demonstrated that the substitution of α88(F9) Ala for a Ser results in increased oxygen affinity and decreased n50 value. The function of the residues involv...
Article
We report on the association of Hb Dunn (alpha 6[A4]Asp----Asn) and Hb O-Arab (beta 121 [GH4]Glu----Lys) in a healthy Moroccan man. Hb Dunn had the same electrophoretic properties as Hb G-Philadelphia, but its percentage was lower. Its identification was based on sequence determination of the alpha T1 peptide. Bgl II and Eco RI mapping showed the p...
Article
We report on the association of Hb Dunn (6[A4]Asp→Asn) and Hb O-Arab (β121[GH4]Glu→Lys) in a healthy Moroccan man. Hb Dunn had the same electrophoretic properties as Hb G-Philadelphia, but its percentage was lower. Its identification was based on sequence determination of the T1 peptide. Bgl II and Eco RI mapping showed the presence of four -genes....