53 results on '"Faouzi Baklouti"'
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2. Proteasome-mediated proteolysis of SRSF5 splicing factor intriguingly co-occurs with SRSF5 mRNA upregulation during late erythroid differentiation.
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Osman Breig and Faouzi Baklouti
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Medicine ,Science - Abstract
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 decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.
- Published
- 2013
- Full Text
- View/download PDF
3. Subtle discrepancies of SF2/ASF ESE sequence motif among human tissues: A computational approach.
- Author
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Olfa Siala, Ahmed Rebai, Faouzi Baklouti, and Faiza Fakhfakh
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- 2010
- Full Text
- View/download PDF
4. The Biochemistry of Survival Motor Neuron Protein Is Paving the Way to Novel Therapies for Spinal Muscle Atrophy
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Olivier Binda, Patrick Lomonte, and Faouzi Baklouti
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business.industry ,animal diseases ,SMN1 ,Motor neuron ,SMA ,Biochemistry ,nervous system diseases ,3. Good health ,medicine.anatomical_structure ,nervous system ,Medicine ,SPINAL MUSCLE ATROPHY ,Treatment strategy ,business ,Gene - Abstract
Spinal muscle atrophy (SMA) is the leading genetic cause of infant mortality. SMA originates from the loss of functional survival motor neuron (SMN) protein. In most SMA cases, the SMN1 gene is deleted. However, in some cases, SMN is mutated, impairing its biological functions. SMN mutants could provide clues about the biological functions of SMN and the specific impact on SMA, potentially leading to the identification of new pathways and thus providing novel treatment alternatives, and even personalized care. Here, we discuss the biochemistry of SMN and the most recent SMA treatment strategies.
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- 2020
5. SMA-linked SMN mutants prevent phase separation properties and SMN interactions with FMRP family members
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Olivier Binda, Franceline Juillard, Julia Novion Ducassou, Constance Kleijwegt, Geneviève Paris, Andréanne Didillon, Faouzi Baklouti, Armelle Corpet, Yohann Couté, Jocelyn Côté, and Patrick Lomonte
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Motor Neurons ,Muscular Atrophy, Spinal ,Proteomics ,Fragile X Mental Retardation Protein ,Ecology ,Health, Toxicology and Mutagenesis ,Humans ,Infant ,RNA ,Plant Science ,Survival of Motor Neuron 1 Protein ,Biochemistry, Genetics and Molecular Biology (miscellaneous) - Abstract
Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of theSMN1gene in most cases or mutations in rare cases. Interestingly, severalSMN1mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that inSMN1mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMRFM). Importantly, SMA-linked SMNTUDORmutant forms (SMNST) failed to interact with FMRFM. In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.
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- 2022
6. The Biochemistry of Survival Motor Neuron Protein Is Paving the Way to Novel Therapies for Spinal Muscle Atrophy
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Patrick, Lomonte, Faouzi, Baklouti, and Olivier, Binda
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Motor Neurons ,Muscular Atrophy, Spinal ,Survival of Motor Neuron 2 Protein ,Mutation ,Animals ,Humans ,Survival of Motor Neuron 1 Protein - Abstract
Spinal muscle atrophy (SMA) is the leading genetic cause of infant mortality. SMA originates from the loss of functional survival motor neuron (SMN) protein. In most SMA cases, the
- Published
- 2020
7. Homozygous deletion of EPB41 genuine AUG-containing exons results in mRNA splicing defects, NMD activation and protein 4.1R complete deficiency in hereditary elliptocytosis
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Madeleine Fénéant-Thibault, Corinne Guitton, Yohann Couté, Faouzi Baklouti, Jean Delaunay, Henri Gruffat, Alain Ninot, Madeleine Morinière, and Amel Haj-Khélil
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RNA Splicing ,Hereditary elliptocytosis ,Molecular Sequence Data ,Erythrocytes, Abnormal ,Biology ,Consanguinity ,Exon ,Elliptocytosis ,medicine ,Humans ,RNA, Messenger ,Allele ,Child ,Peptide Chain Initiation, Translational ,Molecular Biology ,Gene ,Sequence Deletion ,Genetics ,Elliptocytosis, Hereditary ,Membrane Proteins ,EPB41 ,Exons ,Cell Biology ,Hematology ,medicine.disease ,Molecular biology ,Nonsense Mediated mRNA Decay ,Cytoskeletal Proteins ,genomic DNA ,RNA splicing ,Splenectomy ,Molecular Medicine - Abstract
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 a heterozygous 4.1R deficiency with a silent allele. A genomic deletion was found; it encompasses about 50 kb of genomic DNA, and suppresses the two key exons 2 and 4, which contain the two functional AUG translation initiation sites in erythroid and nonerythroid cells. The alternative first exons are intact, hence preserving the transcription potential of the altered gene. Extensive analysis of 4.1R transcripts revealed multiple splicing defects upstream of the deleted sequences. Importantly, we found that most of the transcripts generated from the altered gene are intercepted by the nonsense-mediated mRNA decay mechanism, suggesting that the massive degradation of the mRNA species jeopardizes the production of shortened but functional protein 4.1R from an alternative translation initiation site downstream of the deletion.
- Published
- 2011
8. Xmn I polymorphism associated with concomitant activation of Gγ and Aγ globin gene transcription on a β0-thalassemia chromosome
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Faouzi Baklouti, Amel Haj Khelil, Pascale Perrin, Sandrine Laradi, Madeleine Morinière, Abderrahim Khelif, Jemni Ben Chibani, Laboratoire de biochimie et de biologie moléculaire, Faculté de Pharmacie [Monastir] (FPHM), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
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Reticulocytes ,Transcription, Genetic ,beta-Globins ,0302 clinical medicine ,Transcription (biology) ,MESH: Child ,MESH: Reverse Transcriptase Polymerase Chain Reaction ,hemic and lymphatic diseases ,Gene expression ,Chromosomes, Human ,Child ,Deoxyribonucleases, Type II Site-Specific ,Promoter Regions, Genetic ,Fetal Hemoglobin ,MESH: Deoxyribonucleases, Type II Site-Specific ,MESH: Genetic Association Studies ,MESH: Heterozygote ,Genetics ,0303 health sciences ,MESH: Fetal Hemoglobin ,Reverse Transcriptase Polymerase Chain Reaction ,MESH: beta-Thalassemia ,Hematology ,Pedigree ,Phenotype ,030220 oncology & carcinogenesis ,RNA splicing ,Molecular Medicine ,Female ,MESH: Tunisia ,Adult ,Heterozygote ,Tunisia ,MESH: Mutation ,MESH: Pedigree ,Locus (genetics) ,Biology ,MESH: Phenotype ,MESH: Chromosomes, Human ,MESH: Genetic Loci ,03 medical and health sciences ,alpha-Globins ,MESH: Polymorphism, Genetic ,MESH: Promoter Regions, Genetic ,Fetal hemoglobin ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Molecular Biology ,Genetic Association Studies ,030304 developmental biology ,Polymorphism, Genetic ,MESH: Humans ,MESH: alpha-Globins ,MESH: Transcription, Genetic ,beta-Thalassemia ,Haplotype ,MESH: Adult ,MESH: beta-Globins ,Promoter ,Heterozygote advantage ,MESH: Haplotypes ,Cell Biology ,MESH: Reticulocytes ,Molecular biology ,Haplotypes ,Genetic Loci ,Mutation ,MESH: Female - Abstract
International audience; 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 expression. Interestingly, the β-globin locus haplotype has not been described to bear the (G)γ promoter mutation. Using a gene-specific real-time RT-PCR approach, we found a dramatic increase of both (G)γ and (A)γ mRNA accumulated in the reticulocytes, suggesting that the (G)γ-promoter mutation, alone or in association with another genetic modification, alters in concert the transcription of both (G)γ and (A)γ. This observation is discussed in light of recent regulatory model for β-globin locus.
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- 2011
9. Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop
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Alexandre Douablin, Faouzi Baklouti, Osman Breig, and Orianne Théoleyre
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Cancer Research ,Time Factors ,animal structures ,RNA Splicing ,animal diseases ,Cellular differentiation ,Biology ,Mice ,Phosphatidylinositol 3-Kinases ,Exon ,Erythroid Cells ,Downregulation and upregulation ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Serine ,Genetics ,Animals ,Homeostasis ,Dimethyl Sulfoxide ,Phosphorylation ,Erythropoietin ,Protein Kinase Inhibitors ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Phosphoinositide-3 Kinase Inhibitors ,Regulation of gene expression ,Microfilament Proteins ,Cell Differentiation ,Blood Proteins ,Exons ,Oncogenes ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,Molecular biology ,Globins ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Organ Specificity ,RNA splicing ,Trans-Activators ,bacteria ,Proto-Oncogene Proteins c-akt ,Chromatin immunoprecipitation ,Signal Transduction - Abstract
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 inhibits the erythroid splicing of protein 4.1R exon 16, irrespective of chemical induction of differentiation. Here, we show a positive feedback loop that couples constitutive phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling to high expression of Spi-1/PU.1 in Friend erythroleukemia cells. Inhibition of PI3K/AKT results in Spi-1/PU.1 downregulation in a stepwise manner and induces cell differentiation. Chromatin immunoprecipitation assays further supported the positive autoregulatory effect of Spi-1/PU.1. Mutational analysis indicated that Ser41, but not Ser148, is necessary for Spi-1/PU.1-mediated repression of hemoglobin expression, whereas both Ser residues are required for Spi-1/PU.1 inhibition of the erythroid splicing event. We further show that inhibition of the erythroid transcriptional and splicing events are strictly dependent on distinct Spi-1/PU.1 phosphorylation modifications rather than Spi-1/PU.1 expression level per se. Our data further support the fact that Spi-1/PU.1 inhibits 4.1R erythroid splicing through two different pathways, and bring new insights into the extracellular signal impact triggered by erythropoietin on late erythroid regulatory program, including pre-mRNA splicing.
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- 2010
10. The Hb F composition in a Moroccan Family with β°-thalassaemia and Hb O-Arab
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Jean Delaunay, Jacqueline Godet, François Morlé, Evelyne Dorléac, Claire Baudonnet, L Morle, and Faouzi Baklouti
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Adult ,Proband ,Genetics ,congenital, hereditary, and neonatal diseases and abnormalities ,Hemoglobins, Abnormal ,Cell Membrane ,EcoRI ,Hematology ,Biology ,β thalassaemia ,Phosphoric Monoester Hydrolases ,Pedigree ,Kinetics ,Restriction site ,Normal haemoglobin ,biology.protein ,Humans ,Thalassemia ,Female ,Composition (visual arts) ,Beta (finance) ,Gene ,Fetal Hemoglobin - Abstract
We report on a Moroccan family in which the proposita displays a picture of beta-thalassaemia intermedia, associated with heterozygous Hb O-Arab (beta 121 Glu----Lys) and a beta zero-thalassaemia trait. Hb O-Arab was ascertained by the disappearance of the Eco RI restriction site that normally overlaps the beta-globin gene codon 121. The proposita further presents high proportions of Hb F (12.1%) and of G gamma chains (68.6%). The transmission of the proband's haemoglobin markers was analyzed (the proband's husband displaying normal haemoglobin). The beta zero-thalassaemia and O-Arab genes underwent mutual exclusion. A high Hb F (9.28%) level was found in one child, in association with the beta zero-thalassaemia trait, while another child carrying the latter trait displayed normal levels of Hb F. This situation suggests that a heterocellular HPFH determinant is involved. However, there was no means to establish whether the high Hb F proportion in the mother results solely from the beta zero-thalassaemia -Hb O-Arab association or whether an additional HPFH determinant is present. No DNA deletion was detectably associated with the high proportion of Hb F. In this family, the G gamma percentage was high whenever the beta zero-thalassaemia gene was present, regardless of total Hb F percentage. This observation is consistent with the view that the control of the G gamma percentage in the adult is linked to the beta-locus.
- Published
- 2009
11. Cryptic splicing sites are differentially utilized in vivo
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Jemni Ben Chibani, Mireille Deguillien, Amel Haj Khelil, Faouzi Baklouti, and Madeleine Morinière
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Genetics ,Mutation ,Splice site mutation ,Haplotype ,Intron ,Context (language use) ,Cell Biology ,Biology ,medicine.disease_cause ,Biochemistry ,Exon ,RNA splicing ,medicine ,splice ,Molecular Biology - Abstract
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 contribute to a background lack of fidelity in the context of the wild-type sequence. We also found that a mutation at the 5' splice site of beta-globin intron 1 accommodates multiple cryptic splicing pathways, including three previously reported pathways. Focusing on the two major cryptic 5' splice sites within beta-globin exon 1, we show that cryptic splice site selection ex vivo varies depending upon: (a) the cell stage of development during terminal erythroid differentiation; (b) the nature of the mutation at the authentic 5' splice site; and (c) the nature of the promoter. Finally, we found that the two major cryptic 5' splice sites are utilized with differential efficiencies in two siblings sharing the same beta-globin chromosome haplotype in the homozygous state. Collectively, these data suggest that intrinsic, sequence specific factors and cell genetic background factors both contribute to promote a subtle differential use of cryptic splice sites in vivo.
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- 2008
12. Severe MDC1A Congenital Muscular Dystrophy Due to a Splicing Mutation in theLAMA2Gene Resulting in Exon Skipping and Significant Decrease of mRNA Level
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Chahnez Triki, Pascale Guicheney, Ahmed Rebai, Pascale Richard, Faiza Fakhfakh, Faouzi Baklouti, Olfa Siala, Nacim Louhichi, Madeleine Morinière, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Male ,Genetic Linkage ,RNA Splicing ,Molecular Sequence Data ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,Muscular Dystrophies ,03 medical and health sciences ,Exon ,0302 clinical medicine ,medicine ,Humans ,RNA, Messenger ,Gene ,Genetics (clinical) ,030304 developmental biology ,Genetics ,0303 health sciences ,Messenger RNA ,Base Sequence ,Intron ,Exons ,medicine.disease ,Molecular biology ,Exon skipping ,Open reading frame ,Child, Preschool ,Mutation ,RNA splicing ,Codon, Terminator ,Congenital muscular dystrophy ,Laminin ,RNA Splice Sites ,030217 neurology & neurosurgery - Abstract
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, and DNA sequencing revealed in the propositus of a new homozygous mutation in the donor splice site of intron 58 of the LAMA2 gene. RT-PCR experiments performed on total RNA from a patient's muscle biopsy showed a complete skipping of exon 58 in LAMA2 cDNA and a significant decrease in the LAMA2 mRNA level. This exon skipping altered the open reading frame of the mutant transcript and generated a premature termination codon (PTC) within exon 59, which potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). However, the residual exon 58-lacking mRNA could potentially be translated, and the resulting truncated alpha2 chain would lack its LG4 and LG5 domains that are involved in binding with alpha-dystroglycan. These results demonstrate the utility of mRNA analysis to understand the mutation primary impact and the disease phenotype in the patients.
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- 2007
13. Des oncogènes à l’interface entre transcription et épissage
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Faouzi Baklouti and Orianne Théoleyre
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General Medicine ,Biology ,Transcription factor ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology - Abstract
Le développement de cellules cancéreuses est depuis longtemps associé à des dérèglements de l’activité transcriptionnelle de nombreux gènes, mais aussi, plus récemment, à l’apparition d’anomalies d’épissage des ARN prémessagers. La contribution de ces anomalies d’épissage au développement de cellules tumorales, ainsi que le pouvoir tumorigène de certaines des isoformes protéiques qui en sont issues sont encore très peu explorés. Toutefois, depuis la découverte récente du couplage des deux mécanismes - transcription et épissage - des efforts de recherche ont permis de démontrer que des facteurs de transcription oncogéniques affectent aussi l’épissage des prémessagers. Ces observations suscitent des interrogations quant aux mécanismes d’action des oncogènes à activité transcriptionnelle et, à plus long terme, quant à la recherche de cibles cellulaires nouvelles à appréhender dans de futurs protocoles thérapeutiques anticancéreux., 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 interfere with regulatory cascades that control the growth, differentiation, and survival of normal cells. There is also evidence that alterations of oncogene activity are associated with pre-mRNA splicing defects. The insights gained from the pivotal role of RNA polymerase II in coupling transcription and splicing have instigated a new line of research regarding the possible role of oncogenic transcription factors in pre-mRNA splicing regulation. This review focuses on recent advances addressing this question. Understanding the impact of alterations in the expression and/or function of oncogenes have important prognostic implications that can guide the design of new therapeutic drugs to promote differentiation and/or apoptosis over cell proliferation.
- Published
- 2004
14. Cytoplasmic nonsense-mediated mRNA decay for a nonsense (W262X) transcript of the gene responsible for hereditary tyrosinemia, fumarylacetoacetate hydrolase
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Antonella Maresca, Edward W. Khandjian, Faouzi Baklouti, Robert M. Tanguay, Natacha Dreumont, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
- Subjects
MESH: Mutation ,Hydrolases ,RNA Stability ,Nonsense mutation ,Nonsense-mediated decay ,Biophysics ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,Biochemistry ,Cell Line ,MESH: Tyrosinemias ,Tyrosinemia ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Protein biosynthesis ,Humans ,RNA, Messenger ,MESH: Codon, Nonsense ,Molecular Biology ,Gene ,MESH: RNA, Messenger ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,MESH: Humans ,Tyrosinemias ,MESH: RNA Stability ,Cell Biology ,medicine.disease ,MESH: Gene Expression Regulation ,Molecular biology ,Stop codon ,MESH: Cell Line ,MESH: Hydrolases ,Gene Expression Regulation ,Codon, Nonsense ,Protein Biosynthesis ,MESH: Protein Biosynthesis ,MESH: Subcellular Fractions ,030220 oncology & carcinogenesis ,Mutation ,Fumarylacetoacetate hydrolase ,Subcellular Fractions - Abstract
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 understand the possible impact of NMD on the pathogenesis of hereditary tyrosinemia type I, a severe metabolic disease caused by fumarylacetoacetate hydrolase (FAH) deficiency, we examined the metabolism of FAH mRNA harboring a nonsense mutation, W262X, in lymphoblastoid cell lines derived from patients and their parents. W262X-FAH transcripts show a approximately 20-fold reduction in abundance in mutant cells, which is translation-dependent. Cellular fractionation shows that this down-regulation of the W262X transcript occurs in the cytoplasm. Thus, the W262X FAH is another example of nonsense mRNAs subjected to the NMD pathway in the cytoplasm.
- Published
- 2004
15. A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells
- Author
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Laure Croisille, Faouzi Baklouti, Jean Delaunay, Edward J. Benz, Madeleine Morinière, François Delhommeau, Gabriel Tamagnini, Pierre-Olivier Schischmanoff, Gil Tchernia, Letícia Ribeiro, Patricia Rince, Philippe Leclerc, and Corinne Vasseur-Godbillon
- Subjects
Molecular Sequence Data ,Immunology ,Mitosis ,Spindle Apparatus ,Biology ,medicine.disease_cause ,Biochemistry ,Spindle pole body ,Exon ,Sequence Homology, Nucleic Acid ,RNA Precursors ,medicine ,Humans ,Protein Isoforms ,Amino Acid Sequence ,DNA Primers ,Centrosome ,Mutation ,Base Sequence ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,Neuropeptides ,Membrane Proteins ,Proteins ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Recombinant Proteins ,Spindle apparatus ,Cell biology ,Alternative Splicing ,Cytoskeletal Proteins ,RNA splicing ,Precursor mRNA ,Sequence Alignment ,Cell Division - Abstract
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-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.
- Published
- 2002
16. Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure
- Author
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Gabriel Tamagnini, Letícia Ribeiro, Philippe Maillet, Helena Almeida, Mireille Deguillien, Madeleine Morinière, Jean Delaunay, Thérèse Cynober, François Delhommeau, Nicole Dalla Venezia, Faouzi Baklouti, and Laviron, Nathalie
- Subjects
Adult ,Male ,Vesicle-associated membrane protein 8 ,Erythrocytes ,Protein Conformation ,RNA Splicing ,Hereditary elliptocytosis ,DNA Mutational Analysis ,Molecular Sequence Data ,Immunology ,Biology ,Polymerase Chain Reaction ,Biochemistry ,Structure-Activity Relationship ,Exon ,Elliptocytosis ,Protein structure ,medicine ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,Polymorphism, Single-Stranded Conformational ,Messenger RNA ,C-terminus ,Neuropeptides ,Elliptocytosis, Hereditary ,Protein primary structure ,Membrane Proteins ,Cell Biology ,Hematology ,medicine.disease ,Molecular biology ,Protein Structure, Tertiary ,Molecular Weight ,Cytoskeletal Proteins ,Female - Abstract
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 the 16-kd or the 22/24-kd domain in these cells. We here describe new mutations in the sequence encoding the C-terminal 22/24-kd domain that are associated with hereditary elliptocytosis. An unusually mild phenotype observed in heterozygous and homozygous members of 1 family suggested heterogeneity in the pattern of expression of 4.1R deficiency. Using a variety of protein and messenger RNA (mRNA) quantification strategies, we showed that, regardless of the alteration in the C-terminal primary sequence, when the protein is produced, it assembles at the cell membrane. In addition, we found that alterations in red cell morphologic features and membrane function correlate with the amount of membrane-associated protein-and therefore with the amount of mRNA accumulated-rather than with the primary structure of the variant proteins. These data suggest that an intact sequence at exons 19 through 21 encoding part of the C-terminal 22/24-kd region is not required for proper protein 4.1R assembly in mature red cells. (Blood. 2000;95:1834-1841)
- Published
- 2000
17. Combined inhibition of PI3K and activation of MAPK p38 signaling pathways trigger erythroid alternative splicing switch of 4.1R pre-mRNA in DMSO-induced erythroleukemia cells
- Author
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Faouzi Baklouti, Osman Breig, and Orianne Théoleyre-Schaal
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MAPK/ERK pathway ,Cellular differentiation ,Alternative splicing ,Cell Biology ,Exons ,Biology ,p38 Mitogen-Activated Protein Kinases ,Cell biology ,Exon ,Alternative Splicing ,Mice ,Phosphatidylinositol 3-Kinases ,SR protein ,Erythroid Cells ,Cell Line, Tumor ,RNA splicing ,RNA Precursors ,Animals ,Dimethyl Sulfoxide ,Erythropoiesis ,Leukemia, Erythroblastic, Acute ,Signal transduction ,PI3K/AKT/mTOR pathway ,Phosphoinositide-3 Kinase Inhibitors ,Signal Transduction - Abstract
There is increasing evidence showing that many extracellular cues modulate pre-mRNA alternative splicing, through different signaling pathways. We here show that 4.1R exon 16 splicing is altered in response to specific signals. The switch from erythroblastic isoform lacking exon 16 to mature erythrocytic isoform containing this exon is tightly regulated during late erythroid differentiation, and blocage of this splicing switch in erythroleukemia cells is seen as a consequence of the deregulation of important regulatory pathways. We support that combined inhibition of PI3K and activation of p38 signaling pathways impinge on erythroid 4.1R pre-mRNA alternative splicing switch, and on cell differentiation as witnessed by hemoglobin production. By contrast, MEK/ERK signaling appeared not to affect neither cell hemoglobin production nor erythroid 4.1R pre-mRNA splicing. We also found that the signal-induced alternative splicing is not typically distinctive of EPO-non-responsive cells, but operates in EPO-responsive cells as well. Pre-mRNA splicing is a major regulatory mechanism at the crossroad between transcription and translation. We here provide evidence that inhibition of PI3K activates the splicing switch in a promoter-dependent manner, whereas p38 activation induces this event in a promoter-independent fashion. Our data further support that constitutive activation of EPO-R by the viral protein gp55 and the short form of the tyrosine kinase receptor Stk, transduces PI3K proliferation signal, but not MAPK p38 differentiation signal. Concurrently, this work lend credence to the concept that DMSO triggers transient activation of p38 signaling and irreversible inhibition of PI3K/AKT signaling pathway, hence uncovering an old conundrum regarding the mechanism by which DMSO induces erythroleukemia cell differentiation.
- Published
- 2013
18. Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation
- Author
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Vincent T. Marchesi, Shu-Ching Huang, Jean Delaunay, Edward J. Benz, Faouzi Baklouti, and Tang K. Tang
- Subjects
Protein isoform ,Splice site mutation ,Immunology ,Alternative splicing ,Exonic splicing enhancer ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Exon ,RNA splicing ,Spectrin ,Precursor mRNA - Abstract
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 generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5′ end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD “spectrin/actin binding domain” (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5′ end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.
- Published
- 1996
19. Chimeric probe‐mediated ribonuclease protection assay for molecular diagnosis of mRNA deficiencies
- Author
-
Philippe Maillet, Jean Delaunay, and Faouzi Baklouti
- Subjects
Genetics ,Genetics (clinical) - Published
- 1996
20. Chimeric probe-mediated ribonuclease protection assay for molecular diagnosis of mRNA deficiencies
- Author
-
Faouzi Baklouti, P. Maillet, and Jean Delaunay
- Subjects
Messenger RNA ,Base Sequence ,Transcription, Genetic ,biology ,Erythrocyte Membrane ,Molecular Sequence Data ,Molecular Conformation ,Molecular Probe Techniques ,Single-strand conformation polymorphism ,Nuclease protection assay ,Polymerase Chain Reaction ,Molecular biology ,Cell biology ,Ribonucleases ,Real-time polymerase chain reaction ,Transcription (biology) ,Mechanical stability ,Anion Exchange Protein 1, Erythrocyte ,Genetics ,biology.protein ,Humans ,RNA, Messenger ,Ribonuclease ,Cytoskeleton ,Genetics (clinical) - Abstract
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 bypass fast and commonly used methods, such as SSCP and DGGE, mostly because of a subtle change in mRNA transcription level or a complex interaction leading to the loss of other components. We describe a ribonuclease protection assay based on a simultaneous quantification of two cytoskeletal transcripts, using a chimeric probe, emphasizing the value of a nonspecific bridging sequence, inserted between the two specific probe sequences. It is anticipated that this powerful and reliable procedure would be an additional tool in the methodology array used for screening cytoskeletal inherited abnormalities.
- Published
- 1996
21. Subtle discrepancies of SF2/ASF ESE sequence motif among human tissues: A computational approach
- Author
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Faiza Fakhfakh, Ahmed Rebai, Olfa Siala, Faouzi Baklouti, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Amino Acid Motifs ,Exonic splicing enhancer ,MESH: RNA Splice Sites ,Biology ,Biochemistry ,03 medical and health sciences ,Exon ,MESH: Amino Acid Motifs ,0302 clinical medicine ,SR protein ,Structural Biology ,Humans ,Tissue Distribution ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,MESH: Tissue Distribution ,Conserved Sequence ,030304 developmental biology ,Genetics ,0303 health sciences ,Splice site mutation ,MESH: Conserved Sequence ,MESH: Humans ,Serine-Arginine Splicing Factors ,Organic Chemistry ,Alternative splicing ,Intron ,Computational Biology ,Nuclear Proteins ,RNA-Binding Proteins ,Computational Mathematics ,Enhancer Elements, Genetic ,MESH: RNA-Binding Proteins ,030220 oncology & carcinogenesis ,RNA splicing ,RNA Splice Sites ,MESH: Enhancer Elements, Genetic ,Sequence motif ,MESH: Nuclear Proteins ,MESH: Computational Biology - Abstract
International audience; The intron removal during the pre-mRNA splicing in higher eukaryotes requires the accurate identification of the two splice sites at the ends of the exons, or exon definition. However, the consensus sequences at the splice sites provide insufficient information to distinguish true splice sites from the large number of the false ones that populate the primary transcripts. Additional information is provided by cis-acting regulatory sequences that serve to enhance or repress splicing, and that may be exonic or intronic in nature: the splicing enhancers and the splicing silencers, respectively. In this study, we tested by computational and statistical approaches if the exonic splicing enhancer motif binding to the SF2/ASF SR protein is conserved among several groups of human genes. The results showed that the SF2/ASF ESE consensus was conserved between genes within the same chromosome, within different chromosomes and between different levels of muscular cells differentiation. However, this motif displays subtle variations within the consensus sequence between genes expressed in different tissues. These results can emphasize the presence of different translational isoforms of the SFRS1 gene encoding for the SF2/ASF, or different post-translational protein maturations in different tissues. This tissular discrepancy can also account for the alternative splicing of several genes between tissues.
- Published
- 2010
22. Nonsense-mediated mRNA decay (NMD) blockage promotes nonsense mRNA stabilization in protein 4.1R deficient cells carrying the 4.1R Coimbra variant of hereditary elliptocytosis
- Author
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Jean Delaunay, Faouzi Baklouti, François Delhommeau, Alain Calender, Madeleine Morinière, Letícia Ribeiro, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Léon Bérard [Lyon]-Hospices Civils de Lyon (HCL), Pathologie de la polymérisation des protéines. Substitut du sang et pathologie moléculaire du globule rouge, and Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR93-Université Paris-Sud - Paris 11 (UP11)
- Subjects
MESH: Cytoskeletal Proteins ,MESH: Introns ,Hereditary elliptocytosis ,media_common.quotation_subject ,RNA Stability ,Nonsense-mediated decay ,Nonsense ,Nonsense mutation ,MESH: Elliptocytosis, Hereditary ,MESH: Protein Isoforms ,Biology ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Protein Isoforms ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,MESH: Codon, Nonsense ,RNA, Messenger ,Molecular Biology ,MESH: RNA, Messenger ,030304 developmental biology ,media_common ,0303 health sciences ,Mutation ,Messenger RNA ,MESH: Humans ,MESH: Alternative Splicing ,Elliptocytosis, Hereditary ,Membrane Proteins ,MESH: RNA Stability ,Cell Biology ,Hematology ,MRNA stabilization ,medicine.disease ,Molecular biology ,Introns ,Alternative Splicing ,Cytoskeletal Proteins ,Codon, Nonsense ,RNA splicing ,Molecular Medicine ,MESH: Membrane Proteins ,030217 neurology & neurosurgery - Abstract
International audience; 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 4.1R in the homozygous state in a patient with hereditary elliptocytosis and a moderated hemolytic anemia. We show that, in addition to two known minor shortened and stable spliceoforms, the mutation activates an intronic cryptic splice site, which results in a nonsense mRNA major isoform, targeted to degradation in intact cells by NMD. This accounts for the main cause of 4.1R partial deficiency. In a general perspective, blocking the NMD mechanism would help to identify a missing isoform, and pave the path for a molecular targeting strategy to circumvent a deleterious splicing pathway in favor of a therapeutic splicing pathway.
- Published
- 2010
23. Spectrin Jendouba: an alpha II/31 spectrin variant that is associated with elliptocytosis and carries a mutation distant from the dimer self- association site
- Author
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J Maréchal, L Morle, MT Ducluzeau, R Kastally, C Feo, Nicole Alloisio, L Denoroy, R Wilmotte, Jean Delaunay, Faouzi Baklouti, and BG Forget
- Subjects
Genetics ,chemistry.chemical_classification ,Hereditary elliptocytosis ,Dimer ,Immunology ,Peptide ,macromolecular substances ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Trypsin ,Cleavage (embryo) ,Biochemistry ,Molecular biology ,DNA sequencing ,Elliptocytosis ,chemistry.chemical_compound ,chemistry ,medicine ,Spectrin ,medicine.drug - Abstract
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 responsible mutation is alpha 791 Asp----Glu (GAC----GAA). As in most alpha-spectrin variants associated with elliptocytosis, the change alters helix 3 of the proposed triple helical model of spectrin structure. Modified helix 3 in repeat alpha 8 is the most distant from the N-terminus of alpha-spectrin in known variants associated with elliptocytosis.
- Published
- 1992
24. Elliptocytogenic alpha I/36 spectrin Sfax lacks nine amino acids in helix 3 of repeat 4. Evidence for the activation of a cryptic 5'-splice site in exon 8 of spectrin alpha-gene
- Author
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Jean Delaunay, MH Ben Aribia, Nicole Alloisio, R Kastally, Faouzi Baklouti, MT Ducluzeau, A Mrad, J Maréchal, R Wilmotte, L Morle, and L Denoroy
- Subjects
chemistry.chemical_classification ,Genetics ,Messenger RNA ,Immunology ,Peptide ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Amino acid ,Exon ,chemistry ,RNA splicing ,Spectrin ,splice ,Alpha helix - Abstract
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 maps, the alpha I domain split into two abnormal fragments of 36 and 33 Kd. The mutated alpha-chain represented 20% and 44% of total alpha-chain in alpha/alpha I/36 and alpha V/41/alpha I/36 heterozygotes, respectively. Peptide sequencing showed that the 36-Kd fragment started at Ala 357 and displayed a deletion extending from amino acids 363 to 371. The corresponding 27-nucleotide deletion was found in alpha-spectrin mRNA. However, exon 8 of spectrin alpha-gene failed to disclose this deletion. Instead, an A to G substitution appeared in position 3 of codon 362, leading to the occurrence of the critical GU dinucleotide within a cryptic 5′-splice site surrounding codon 362. This event would account for the splicing out of codons 363 to 371. The reading frame was preserved and even amino acid 362 (AGG, Arg) remained unaltered. As in most spectrin alpha-chain elliptocytogenic variants, the change involved a helix 3. This is the first elliptocytogenic mutation recorded in repeat alpha 4.
- Published
- 1992
25. Molecular analysis of hereditary elliptocytosis with reduced protein 4.1 in the French Northern Alps
- Author
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Edward J. Benz, L Morle, S Chabanis, Jean Delaunay, J. M. Robert, S Feddal, J Maréchal, MT Ducluzeau, G. Brunet, L Roda, Nicole Alloisio, and Faouzi Baklouti
- Subjects
Genetics ,Messenger RNA ,Hereditary elliptocytosis ,Haplotype ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Loss of heterozygosity ,Elliptocytosis ,Complementary DNA ,medicine ,Coding region ,Northern blot - Abstract
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. They were recruited independently (to the exclusion of any other variety of HE) in a limited area around the city of Annecy (French Northern Alps). The mode of genetic transmission, as well as the clinical, morphologic, and protein phenotypes fully conformed to the classical description. Western blots ruled out the existence of any protein 4.1 species of abnormal size. No obvious DNA rearrangement was detectable in any of the nine families with three 4.1 cDNA probes covering the entire coding sequence and part of the flanking 5′ and 3′ untranslated sequences. On the basis of five polymorphic sites (Bgl II, 2; Pvu II, 3), we found five different haplotypes in normal members of the 4.1(-) families. 4.1(-) HE was associated with the most common haplotype in all the propositi. 4.1 mRNA was studied in four families. Dot-blot hybridization experiments and Northern blots failed to show any detectable change in three families. On the other hand, they showed a 2-kb deletion in the 4.1(-) messenger RNA 5′-moiety in one family. These findings emphasize the heterogeneity of 4.1(-) HE at the molecular level.
- Published
- 1991
26. Downregulation of the Spi-1/PU.1 oncogene induces the expression of TRIM10/HERF1, a key factor required for terminal erythroid cell differentiation and survival
- Author
-
Rand Blaybel, Faouzi Baklouti, Orianne Théoleyre, and Alexandre Douablin
- Subjects
Erythrocytes ,Cell Survival ,RNA Splicing ,Down-Regulation ,Biology ,Tripartite Motif Proteins ,Exon ,Hemoglobins ,Mice ,Downregulation and upregulation ,Erythroid Cells ,Transcription (biology) ,Cell Line, Tumor ,Histocompatibility Antigens ,Proto-Oncogene Proteins ,Gene silencing ,Animals ,Dimethyl Sulfoxide ,Molecular Biology ,Messenger RNA ,Gene knockdown ,Oncogene ,Proto-Oncogene Protein c-fli-1 ,Microfilament Proteins ,Intracellular Signaling Peptides and Proteins ,Cell Differentiation ,Cell Biology ,Blood Proteins ,Exons ,Molecular biology ,Hematopoiesis ,Up-Regulation ,RNA splicing ,Trans-Activators ,RNA Interference ,Carrier Proteins ,Signal Transduction - Abstract
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 demonstrates that Spi-1/PU.1 downregulation induces the activation of TRIM10/hematopoietic RING finger 1 (HERF1), a member of the tripartite motif (TRIM)/RBCC protein family needed for globin gene transcription. Additionally, we demonstrate that TRIM10/HERF1 is required for the regulated splicing of exon 16 during late erythroid differentiation. Using inducible overexpression and silencing approaches, we found that: (1) TRIM10/HERF1 knockdown inhibits hemoglobin production and exon splicing and triggers cell apoptosis in dimethylsulfoxide (DMSO)-induced cells; (2) TRIM10/HERF1 upregulation is required but is insufficient on its own to activate exon retention; (3) Fli-1 has no effect on TRIM10/HERF1 expression, whereas either DMSO-induced downregulation or shRNA-knockdown of Spi-1/PU.1 expression is sufficient to activate TRIM10/HERF1 expression; and (4) Spi-1/PU.1 knockdown triggers both the transcription and the splicing events independently of the chemical induction. Altogether, these data indicate that primary Spi-1/PU.1 downregulation acts on late erythroid differentiation through at least two pathways, one of which requires TRIM10/HERF1 upregulation and parallels the Spi-1/PU.1-induced Fli-1 shutoff regulatory cascade.
- Published
- 2008
27. Cryptic splicing sites are differentially utilized in vivo
- Author
-
Amel, Haj Khelil, Mireille, Deguillien, Madeleine, Morinière, Jemni, Ben Chibani, and Faouzi, Baklouti
- Subjects
Transcription, Genetic ,Mutation ,Humans ,Exons ,RNA Splice Sites ,Promoter Regions, Genetic ,Cells, Cultured ,Introns ,Globins - Abstract
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 contribute to a background lack of fidelity in the context of the wild-type sequence. We also found that a mutation at the 5' splice site of beta-globin intron 1 accommodates multiple cryptic splicing pathways, including three previously reported pathways. Focusing on the two major cryptic 5' splice sites within beta-globin exon 1, we show that cryptic splice site selection ex vivo varies depending upon: (a) the cell stage of development during terminal erythroid differentiation; (b) the nature of the mutation at the authentic 5' splice site; and (c) the nature of the promoter. Finally, we found that the two major cryptic 5' splice sites are utilized with differential efficiencies in two siblings sharing the same beta-globin chromosome haplotype in the homozygous state. Collectively, these data suggest that intrinsic, sequence specific factors and cell genetic background factors both contribute to promote a subtle differential use of cryptic splice sites in vivo.
- Published
- 2008
28. LAMA2 mRNA processing alterations generate a complete deficiency of laminin-alpha2 protein and a severe congenital muscular dystrophy
- Author
-
Faiza Fakhfakh, Madeleine Morinière, Chahnez Triki, Nacim Louhichi, Olfa Siala, and Faouzi Baklouti
- Subjects
Male ,Genetic Linkage ,RNA Splicing ,Nonsense-mediated decay ,Biology ,Bioinformatics ,Transfection ,Severity of Illness Index ,Muscular Dystrophies ,Frameshift mutation ,Exon ,Fatal Outcome ,medicine ,Humans ,RNA, Messenger ,Child ,Gene ,Genetics (clinical) ,Polymorphism, Single-Stranded Conformational ,Genetics ,Intron ,Single-strand conformation polymorphism ,medicine.disease ,Introns ,Pedigree ,Phenotype ,Neurology ,Pediatrics, Perinatology and Child Health ,RNA splicing ,Congenital muscular dystrophy ,Female ,Neurology (clinical) ,Laminin ,Gene Deletion ,HeLa Cells - Abstract
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 lumbar scoliosis and respiratory complications in a patient, who died at the age of 10. Despite a poor linkage to any form of CMD, total deficiency of laminin-α2 rather suggested the occurrence of an MDC1A form. Extensive analysis of LAMA2 gene revealed two novel mutations: a (8007delT) frameshift deletion in exon 57, and a de novo 7 nt deletion in intron 17. Using an ex vivo approach, we provided strong evidence that the intron mutation is responsible for complete exon 17 skipping. The mutations are in trans and they each generate a nonsense mRNA potentially elicited to degradation by NMD. We further discuss the impact of mRNA alterations on the subtle phenotypic discrepancies.
- Published
- 2007
29. Protein 4.1R expression in normal and dystrophic skeletal muscle
- Author
-
François Delhommeau, Philippe Maillet, Madeleine Morinière, Jean Delaunay, Philippe Leclerc, Ibtissem Guerfali, Michel Fardeau, Nicole Dalla Venezia, Faouzi Baklouti, H. Collin, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, and Laviron, Nathalie
- Subjects
MESH: Cytoskeletal Proteins ,Duchenne muscular dystrophy ,MESH: Amino Acid Sequence ,MESH: Base Sequence ,Muscular Dystrophies ,MESH: Recombinant Proteins ,Exon ,MESH: Reverse Transcriptase Polymerase Chain Reaction ,RNA Precursors ,MESH: Blood Proteins ,Muscular dystrophy ,MESH: Muscle, Skeletal ,Myogenesis ,Reverse Transcriptase Polymerase Chain Reaction ,MESH: Alternative Splicing ,General Medicine ,Blood Proteins ,Recombinant Proteins ,Cell biology ,medicine.anatomical_structure ,MESH: Membrane Proteins ,General Agricultural and Biological Sciences ,Dystrophin ,Microtubule-Associated Proteins ,medicine.medical_specialty ,DNA, Complementary ,Molecular Sequence Data ,MESH: RNA Precursors ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Internal medicine ,medicine ,Humans ,Amino Acid Sequence ,Muscle, Skeletal ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,Sarcolemma ,MESH: Molecular Sequence Data ,MESH: Humans ,General Immunology and Microbiology ,Base Sequence ,Alternative splicing ,Skeletal muscle ,Membrane Proteins ,MESH: DNA, Complementary ,medicine.disease ,MESH: Muscular Dystrophies ,MESH: Cell Line ,Alternative Splicing ,Cytoskeletal Proteins ,MESH: Microtubule-Associated Proteins ,Endocrinology ,biology.protein - Abstract
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 epitopes are also found at the sarcolemma of both slow and fast myofibres in normal muscle. Interestingly, they are absent from dystrophin-deficient sarcolemma of DMD muscle, and colocalize with partially expressed dystrophin in BMD muscle. We also show that alternative splicing of exons 16 and 17a is regulated during muscle differentiation in an asynchronous fashion, with an early inclusion of exon 16 in forming myotubes, and a late inclusion of exon 17a. Consistently, Western blot analysis led to characterize mainly an approximately 96/98-kDa doublet bearing exons 16-17a-encoding peptide, exclusively occurring in the differentiated muscle.
- Published
- 2005
30. [Oncogenic transcription factors as splicing regulators]
- Author
-
Orianne, Théoleyre and Faouzi, Baklouti
- Subjects
Cell Transformation, Neoplastic ,Gene Expression Regulation ,RNA Splicing ,Humans ,Apoptosis ,Cell Differentiation ,Oncogenes ,Signal Transduction ,Transcription Factors - Abstract
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 interfere with regulatory cascades that control the growth, differentiation, and survival of normal cells. There is also evidence that alterations of oncogene activity are associated with pre-mRNA splicing defects. The insights gained from the pivotal role of RNA polymerase II in coupling transcription and splicing have instigated a new line of research regarding the possible role of oncogenic transcription factors in pre-mRNA splicing regulation. This review focuses on recent advances addressing this question. Understanding the impact of alterations in the expression and/or function of oncogenes have important prognostic implications that can guide the design of new therapeutic drugs to promote differentiation and/or apoptosis over cell proliferation.
- Published
- 2004
31. Spi-1/PU.1 but not Fli-1 inhibits erythroid-specific alternative splicing of 4.1R pre-mRNA in murine erythroleukemia cells
- Author
-
Françoise Moreau-Gachelin, Orianne Théoleyre, François Morlé, Mireille Deguillien, Madeleine Morinière, Joëlle Starck, Faouzi Baklouti, and Laviron, Nathalie
- Subjects
Cancer Research ,Cellular differentiation ,Biology ,Exon ,Mice ,hemic and lymphatic diseases ,Proto-Oncogene Proteins ,Genetics ,RNA Precursors ,Tumor Cells, Cultured ,Animals ,RNA, Messenger ,Molecular Biology ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,DNA Primers ,Messenger RNA ,Base Sequence ,Proto-Oncogene Protein c-fli-1 ,Alternative splicing ,Cell Differentiation ,Transfection ,Exons ,Molecular biology ,DNA-Binding Proteins ,Alternative Splicing ,RNA splicing ,Trans-Activators ,Leukemia, Erythroblastic, Acute ,Precursor mRNA ,Differentiation Inducer - Abstract
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 erythroleukemic cells that have the potential to differentiate and produce hemoglobin, regardless of the nature of the differentiation inducer. Knowing that dysregulated expression of spi-1/pu.1 and fli-1 oncogenes is involved in MEL cell differentiation arrest, we looked at their effect on exon 16 erythroid splicing. We found that exon 16 inclusion requires Spi-1/PU.1 shutdown in MEL cells, and that enforced expression of Spi-1/PU.1 inhibits exon selection, regardless of the presence or absence of a chemical inducer. By contrast, endogenous overexpression or enforced expression of Fli-1 has no effect on exon selection. We further showed that Spi-1/PU.1 acts similarly on the endogenous and on a transfected exon 16, suggesting a promoter-independent effect of Spi-1/PU.1 on splicing regulation. This study provides the first evidence that Spi-1/PU.1 displays the unique property, not shared with Fli-1, to inhibit erythroid-specific pre-mRNA splicing in erythroleukemia cell context.
- Published
- 2004
32. JAK2V617F mRNA metabolism in myeloproliferative neoplasm cell lines
- Author
-
Faouzi Baklouti, François Delhommeau, and P Nauroy
- Subjects
RNA Splicing ,Myeloproliferative Disorders ,Myeloid stem cell ,Polycythemia vera ,hemic and lymphatic diseases ,Cell Line, Tumor ,medicine ,Humans ,RNA, Messenger ,Myelofibrosis ,Letter to the Editor ,Myeloproliferative neoplasm ,Janus kinase 2 ,biology ,Essential thrombocythemia ,Myeloid leukemia ,Hematology ,Janus Kinase 2 ,medicine.disease ,Oncology ,Haplotypes ,Immunology ,Mutation ,biology.protein ,Cancer research - Abstract
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of leukemias with defective regulation of myeloid stem cell proliferation. They include four distinct diseases: chronic myeloid leukemia, polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).1, 2 In 2005, four independent studies have concurred to the identification in MPN patients of a specific mutation in the Janus kinase 2 (JAK2) protein (1849 G→T in exon 14; 617Val→Phe) in the majority of MPNs.1, 2 Indeed, this JAK2V617F variant was identified in 95% of PV, 55% of ET and 65% of PMF patients. The amino-acid change triggers a constitutive activation of the JAK2 protein, independently of cytokines. Whether JAK2V617F is the initiating event in these MPNs remains under debate; however, JAK2V617F seems to drive the phenotype of the disease.
- Published
- 2014
33. Multiple cis elements regulate an alternative splicing event at 4.1R pre-mRNA during erythroid differentiation
- Author
-
Edward J. Benz, Shu-Ching Huang, Faouzi Baklouti, Madeleine Morinière, Mireille Deguillien, and Natacha Dreumont
- Subjects
Immunology ,Molecular Sequence Data ,Exonic splicing enhancer ,Biology ,Regulatory Sequences, Nucleic Acid ,Exon shuffling ,Transfection ,Biochemistry ,Exon ,Mice ,RNA Precursors ,Tumor Cells, Cultured ,Animals ,Humans ,RNA, Messenger ,Erythroid Precursor Cells ,Splice site mutation ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Alternative splicing ,Neuropeptides ,Intron ,Membrane Proteins ,Proteins ,Cell Differentiation ,Cell Biology ,Hematology ,Exons ,Sequence Analysis, DNA ,Molecular biology ,Introns ,Alternative Splicing ,Cytoskeletal Proteins ,Enhancer Elements, Genetic ,RNA splicing ,Mutation ,Leukemia, Erythroblastic, Acute ,Minigene - Abstract
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 erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5′ splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5′ splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16. The activation of the enhancer elements in late erythroid differentiation may play an important role in the retention of exon 16.
- Published
- 2001
34. A premature termination codon within an alternative exon affecting only the metabolism of transcripts that retain this exon
- Author
-
Faouzi Baklouti, Bernard Noël, Bozon M, Philippe Maillet, Madeleine Morinière, N Dalla Venezia, F Lorenzo, and J Delaunay
- Subjects
Silent mutation ,Genetic Markers ,Male ,Erythrocytes ,Transcription, Genetic ,Nonsense mutation ,DNA Mutational Analysis ,Biology ,Exon shuffling ,Exon ,Exon trapping ,Genetics ,RNA Precursors ,Humans ,Frameshift Mutation ,Genetics (clinical) ,Sequence Deletion ,Splice site mutation ,Polymorphism, Genetic ,Reverse Transcriptase Polymerase Chain Reaction ,Alternative splicing ,Neuropeptides ,Elliptocytosis, Hereditary ,Membrane Proteins ,Cell Differentiation ,Exons ,Molecular biology ,Pedigree ,Open reading frame ,Alternative Splicing ,Cytoskeletal Proteins ,Gene Expression Regulation ,Codon, Nonsense ,Codon, Terminator ,Female ,France - Abstract
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 a single nucleotide deletion of the erythroid protein 4.1 gene associated with hereditary elliptocytosis. The deletion located in exon 16 leads to a frameshift and a premature termination codon within the same exon. In an effort to examine the premature stop codon effect in relationship with exon 16 alternative splicing, we analyzed erythroid and lymphoid protein 4.1 mRNAs using the mutation and a linked downstream polymorphism as markers. We found that the premature stop codon does not affect the tissue-specific alternative splicing among the two cell types analyzed and that the resulting alteration of mRNA metabolism correlates with the retention of exon 16 in reticulocytes. Conversely, skipping of exon 16 in lymphoid cells converts the mutant mRNA to a normal lymphoid-specific mRNA isoform, hence bypassing the nonsense codon. Consistent with data obtained on constitutive nonsense exons, our observations argue in favor of a stop codon recognition mechanism that occurs after the regulated splicing status of the nonsense exon has been achieved.
- Published
- 1999
35. Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA
- Author
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Jean Delaunay, Shu-Ching Huang, Edward J. Benz, Faouzi Baklouti, and Tom Vulliamy
- Subjects
Untranslated region ,Gene isoform ,DNA, Complementary ,Mature messenger RNA ,Molecular Sequence Data ,Biology ,Exon ,Mice ,Genetics ,RNA Precursors ,Coding region ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Binding Sites ,Base Sequence ,Alternative splicing ,Neuropeptides ,Intron ,Chromosome Mapping ,Membrane Proteins ,Spectrin ,Exons ,Actins ,Introns ,Rats ,Alternative Splicing ,Cytoskeletal Proteins ,Protein Biosynthesis ,RNA splicing - Abstract
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 tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3'-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3' UTR, over 3 kb, accounts for one-half of the mature mRNA.
- Published
- 1997
36. Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation
- Author
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Faouzi Baklouti and Osman Breig
- Subjects
Cellular differentiation ,Red Cells ,Gene Expression ,lcsh:Medicine ,Gene Splicing ,Mice ,Molecular Cell Biology ,Phosphorylation ,lcsh:Science ,Erythroid Precursor Cells ,Multidisciplinary ,Serine-Arginine Splicing Factors ,RNA-Binding Proteins ,Cell Differentiation ,Hematology ,Protein-Tyrosine Kinases ,Protein Transport ,RNA splicing ,Medicine ,Signal transduction ,Research Article ,Proteasome Endopeptidase Complex ,RNA Splicing ,Molecular Sequence Data ,Protein Serine-Threonine Kinases ,Biology ,Cell Line ,Molecular Genetics ,Splicing factor ,SR protein ,Erythroid Cells ,Genetics ,Animals ,Protein Interaction Domains and Motifs ,RNA, Messenger ,Protein kinase B ,Base Sequence ,lcsh:R ,Alternative splicing ,Computational Biology ,Molecular biology ,Hematopoiesis ,Gene Expression Regulation ,Proteasome ,Proteolysis ,lcsh:Q ,Protein Processing, Post-Translational ,Proto-Oncogene Proteins c-akt ,Sequence Alignment ,Developmental Biology - Abstract
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 decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.
- Published
- 2013
37. Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position
- Author
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G. Fermi, Josee Pagnier, Yue Huang, Claude Poyart, Faouzi Baklouti, Philippe Magne, Jean Delaunay, Max F. Perutz, and Jean Kister
- Subjects
Binding Sites ,Chemistry ,Stereochemistry ,Protein Conformation ,Hemoglobins, Abnormal ,Mutant ,Hemoglobin variants ,Mutagenesis (molecular biology technique) ,Genetic Variation ,Biochemistry ,Oxygen ,Structure-Activity Relationship ,Protein structure ,X-Ray Diffraction ,Mutation ,Humans ,Hemoglobin ,Binding site ,Beta (finance) ,Protein secondary structure - Abstract
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 relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.
- Published
- 1990
38. [Untitled]
- Author
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Natacha Dreumont, Harvey Louis Levy, Jacques Poudrier, Anne Bergeron, Faouzi Baklouti, and Robert M. Tanguay
- Subjects
Genetics ,Nonsense mutation ,Biology ,medicine.disease ,Tyrosinemia Type I ,Molecular biology ,Tyrosinemia ,Exon ,Mutation (genetic algorithm) ,RNA splicing ,medicine ,Missense mutation ,Fumarylacetoacetate hydrolase ,Genetics (clinical) - Abstract
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 expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules. Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in non-expressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment. These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells.
- Published
- 2001
39. Severe MDC1A Congenital Muscular Dystrophy Due to a Splicing Mutation in the LAMA2Gene Resulting in Exon Skipping and Significant Decrease of mRNA Level.
- Author
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Olfa Siala, Nacim Louhichi, Chahnez Triki, Madeleine Morinière, Ahmed Rebai, Pascale Richard, Pascale Guicheney, Faouzi Baklouti, and Faiza Fakhfakh
- Published
- 2007
- Full Text
- View/download PDF
40. Increased oxygen affinity with normal heterotropic effects in hemoglobin Loire [α88(F9)Ala→Ser]
- Author
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Georges Teyssier, Henri Wajcman, Jean Delaunay, Jean Kister, Faouzi Baklouti, V. Baudin-Chich, Claude Poyart, and Michael C. Marden
- Subjects
Hemeprotein ,Bezafibrate ,Chemistry ,Stereochemistry ,Increased oxygen affinity ,Oxygene ,chemistry.chemical_element ,Bohr effect ,Biochemistry ,Oxygen ,medicine ,Hemoglobin ,computer ,Oxygen binding ,computer.programming_language ,medicine.drug - Abstract
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 involved in the Bohr effect or in the regulation by 2,3-bisphosphoglycerate is not altered. The effects of bezafibrate, which binds specifically to the α chains, was similar to that observed in Hb A. The functional properties of Hb Loire may be explained by a slight displacement of some key residues of the C-terminal region of the α chain destabilizing the T structure.
- Published
- 1988
41. The Association of Hemoglobin Knossos and Hemoglobin Lepore in an Algerian Patient
- Author
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C. Baudonnet, L. Morle, Jacques Delaunay, Faouzi Baklouti, Jacqueline Godet, E. Dorléac, and François Morlé
- Subjects
Male ,medicine.medical_specialty ,P50 ,Hemoglobins, Abnormal ,Clinical Biochemistry ,Biology ,Hb Knossos ,Internal medicine ,medicine ,Humans ,Hemoglobin Knossos ,Genetics (clinical) ,Genetics ,Biochemistry (medical) ,Hemoglobin variants ,Hematology ,Middle Aged ,Total hemoglobin ,Endocrinology ,Hemoglobin Lepore ,Male patient ,Algeria ,Splenectomy ,Thalassemia ,France ,Isoelectric Focusing - Abstract
We report on a 54 years-old male patient from North-Eastern Algeria who combines two hemoglobin variants that are associated with thalassemia-like disorders: Hb Lepore and Knossos (beta 27 Ala----Ser) (1, 2). A beta-thalassemia intermedia picture gradually developed and finally required splenectomy at the age of 53. Total absence of Hb A2 indicated that the beta Knossos gene is most probably flanked with a delta(0)-thalassemia gene. No DNA deletion additional to the Lepore deletion was found. Hb F was elevated (12.3%) with 24% G gamma Hb F. In whole cells, Hb Knossos, representing 70% of total hemoglobin, displayed a decreased affinity for oxygen (P50 = 35 mm Hg), a fact presumably accounting for the relatively good tolerance of the condition.
- Published
- 1984
42. Hehemoglobin Pierre-Bénite [β90(F6)Glu→Asp], a new High Affinity Variant Found in a French Family
- Author
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Faouzi Baklouti, Alain Francina, Jean Delaunay, J. Favre-Gilly, Y. Giraud, and G. Richard
- Subjects
Adult ,Male ,P50 ,Stereochemistry ,Hemoglobins, Abnormal ,Molecular Sequence Data ,Clinical Biochemistry ,chemistry.chemical_element ,Polycythemia ,medicine.disease_cause ,High-performance liquid chromatography ,Oxygen ,Amino acid analysis ,medicine ,Humans ,Amino Acid Sequence ,Chromatography, High Pressure Liquid ,Genetics (clinical) ,Mutation ,Chromatography ,Chemistry ,Biochemistry (medical) ,Tryptic peptide ,Hematology ,Globins ,Hemoglobinopathies ,Electrophoresis ,Hemoglobin - Abstract
Hemoglobin Pierre-Bénite [beta 90(F6)Glu---Asp] is a new high affinity variant (P50 = 21.5 mm Hg), with normal heme-heme interaction, found in a French family. It was difficult to detect by conventional electrophoretic methods. However, the high performance liquid chromatography profile of its tryptic peptides contained an additional peak. Amino acid analysis of the corresponding peptide and determination of its sequence allowed us to identify the mutation. No instability was found. Mutations previously recorded in position 90 of the beta-chain display a positive charge shift and a reduced affinity for oxygen, whereas Hb Pierre-Bénite shows no charge shift and increased affinity for oxygen.
- Published
- 1988
43. Hb Lepore-Hb C and Hb Lepore-β°-Thalassemia Compound Heterozygotes1 N an Algerian Family
- Author
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L Roda, E. Dorléac, Jacques Delaunay, Faouzi Baklouti, Elwan S, N Phillipe, M Aubry, and Alain Francina
- Subjects
Hemoglobin C ,Thalassemia ,Biochemistry (medical) ,Clinical Biochemistry ,medicine ,Heterozygote advantage ,Hematology ,Biology ,medicine.disease ,Beta (finance) ,Compound heterozygosity ,Molecular biology ,Genetics (clinical) - Published
- 1985
44. Hemoglobin Knossos [α2β227(B9)Ala →] in Egypt
- Author
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Jean Delaunay, Faouzi Baklouti, Elwan S, el-Kabsh M, and Abdelrahman F
- Subjects
Biochemistry (medical) ,Clinical Biochemistry ,Genetic variants ,Hematology ,Biology ,medicine.disease ,Molecular biology ,Hemoglobinopathy ,mental disorders ,medicine ,Beta (finance) ,Hemoglobin Knossos ,psychological phenomena and processes ,Genetics (clinical) - Abstract
L'hemoglobine Knossos est un variant β-thalassemique caracterise par la substitution de Ala→SeR en position 27
- Published
- 1987
45. A new Case of Hb Little Rock [β143(H21)His → Gln], a high affinity variant. Study during pregnancy
- Author
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C. Lacombe, Frédéric Galactéros, Alain Francina, Faouzi Baklouti, Jacques Delaunay, G. Souillet, E. Dorléac, Nicole Arous, R. C. Rudigoz, and Jean-Philippe Rosa
- Subjects
Adult ,medicine.medical_specialty ,P50 ,Hemoglobins, Abnormal ,Clinical Biochemistry ,chemistry.chemical_element ,Peptide ,Cooperativity ,Biology ,Oxygen ,High-performance liquid chromatography ,Pregnancy ,Internal medicine ,medicine ,Humans ,Amino Acids ,Chromatography, High Pressure Liquid ,Genetics (clinical) ,chemistry.chemical_classification ,Fetus ,Biochemistry (medical) ,Hematology ,medicine.disease ,Endocrinology ,chemistry ,Gestation ,Female - Abstract
A new case of Hb Little Rock [beta 143(H21)His----Gln] is described. This high affinity variant (P50 = 15 mm Hg in whole cells) has a nearly normal cooperativity. The abnormal beta chain is readily detectable using urea-Triton X-100 electrophoresis. The altered beta T-14 peptide was separated by high performance liquid chromatography. The proposita was followed during a pregnancy. Oxygen unloading was reduced, but normal oxygen loading was maintained in the fetus. There were neither placental abnormalities, nor intrauterine growth retardation.
- Published
- 1987
46. Hemoglobin Grange-Blanche [β27(B9) Ala → Val], a new variant with normal expression and increased affinity for oxygen
- Author
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G. Richard, Jean Delaunay, C. Périer, J. Jaubert, A. Geyssant, Alain Francina, Y. Giraud, C. Brizard, and Faouzi Baklouti
- Subjects
Adult ,Hemeprotein ,P50 ,Protein Conformation ,Hemoglobins, Abnormal ,Biophysics ,Globin chain variant ,Heme ,Biochemistry ,Protein structure ,Structural Biology ,Peptide mapping ,Genetics ,Humans ,Amino Acid Sequence ,Beta (finance) ,Molecular Biology ,Polyacrylamide gel electrophoresis ,Peptide sequence ,Chromatography, High Pressure Liquid ,Chemistry ,Isoelectric focusing ,Cell Biology ,Oxygen affinity ,body regions ,Oxyhemoglobins ,Female ,Hemoglobin - Abstract
Hemoglobin Grange-Blanche [beta 27(B9) Ala----Val] is a new variant found in a Portuguese family. The carriers present moderate erythrocytosis. Upon isoelectric focusing, Hb Grange-Blanche was slightly more cathodic than Hb A. beta Grange-Blanche chain migrated like the G gamma chain when submitted to electrophoresis in the presence of urea-Triton X-100. The precentage of Hb Grange-Blanche was about 50% in the heterozygous state. Oxygen affinity was increased (P50 = 22 mmHg), but heme-heme interaction was normal. An abnormal tryptic peptide (beta T3) was isolated using HPLC. Its composition allowed us to deduce unambiguously the amino acid change. The latter is the third mutation found in position 27 of the beta-chain. Because of its normal expression and its elevated affinity for oxygen, Hb Grange-Blanche contrasts with Hb Knossos [beta 27(B9) Ala----Ser], a beta-thalassemic variant with low affinity.
- Full Text
- View/download PDF
47. δ°-Thalassemia in cis of βKnossos-globin gene Normal structure and normal transient expression of the δ-globin gene
- Author
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Colette Gonnet, Jean Delaunay, Faouzi Baklouti, D. Bozon, J Godet, and R. Ouazana
- Subjects
Regulation of gene expression ,Genetics ,COS cells ,Expression vector ,Biophysics ,Cell Biology ,Transient expression ,Biology ,Biochemistry ,Molecular biology ,chemistry.chemical_compound ,chemistry ,Structural Biology ,Transcription (biology) ,Gene expression ,Gene, δ-globin ,Thalassemia ,Globin ,Molecular Biology ,Gene ,DNA ,DNA sequence polymorphism - Abstract
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. Only two nucleotide changes were recorded: a C → T substitution at −199 and an AT insertion at −448 upstream from the cap site. To examine the involvement of these changes in gene function, the δ-gene was subcloned in an expression vector and introduced into COS cells. Analysis of RNA derived from these cells, using an S1 protection assay and dot-blot hybridization, revealed qualitatively and quantitatively normal transcription. The loss of δ-globin gene activity in vivo may be due to the alteration of a tissue-specific control.
- Full Text
- View/download PDF
48. Hemoglobin J-Baltimore (beta 16(A13)Gly----Asp): interference with the assay of HbA1c
- Author
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A. Bertrand, F. Vandenesch, C. Vianey-Liaud, Faouzi Baklouti, Jean Delaunay, C. Le Dévéhat, and Alain Francina
- Subjects
Adult ,Male ,Glycosylation ,Hemoglobins, Abnormal ,Clinical Biochemistry ,Biochemistry ,High-performance liquid chromatography ,chemistry.chemical_compound ,Large peak ,Diabetes Mellitus ,Humans ,False Positive Reactions ,Hemoglobin J Baltimore ,Chromatography, High Pressure Liquid ,Glycated Hemoglobin ,Hemoglobin J ,Chromatography ,Isoelectric focusing ,Biochemistry (medical) ,Tryptic peptide ,Hemoglobin variants ,General Medicine ,Middle Aged ,chemistry ,Hemoglobin ,Isoelectric Focusing - Abstract
Three independent cases of Hemoglobin J-Baltimore(beta 16(A13)Gly----Asp) were detected through the assay of HbA1c in diabetic patients. Using chromatography on Bio-Rex 70 resin, one large peak replaced the usually well resolved peaks of HbA1a + b and HbA1c. The species that overlapped the latter fractions was identified as HbJ1c. HbJ-Baltimore itself was identified using HPLC of the beta-chain tryptic peptides. This observation emphasizes the errors that hemoglobin variants may introduce in the assay of HbA1c.
- Published
- 1987
49. Association in cis of beta +-thalassemia and hemoglobin S
- Author
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Jean Delaunay, Faouzi Baklouti, Alain Francina, Georges Richard, Evelyne Dorléac, J Godet, and Daniel Rosenberg
- Subjects
Adult ,Male ,Thalassemia ,Hemoglobin, Sickle ,Peptide ,Biology ,hemic and lymphatic diseases ,medicine ,Humans ,Beta (finance) ,Codon ,chemistry.chemical_classification ,Base Sequence ,Microcytosis ,Hemoglobin variants ,Beta thalassemia ,Genetic Variation ,Hemoglobin A ,Hematology ,medicine.disease ,Molecular biology ,Pedigree ,Hemoglobinopathy ,chemistry ,Child, Preschool ,Female ,Hemoglobin - Abstract
A Moroccan woman was investigated because of a typical beta-thalassemia trait associated with a low-percentage (11%) hemoglobin (Hb) variant. The beta-thalassemia trait was manifested by a microcytosis, a high HbA2 (above 6%), and an increase of the alpha/beta biosynthetic ratio (1.31). The variant was identified to HbS by amino acid analysis of the abnormal peptide (beta T1) and by DNA mapping with Sau I (Mst II) restriction endonuclease. No additional amino acid substitution was recorded in the beta s-chain. The reduction of beta-globin synthesis occurred exclusively at the expense of the beta s-chain. These results are consistent with the existence of a beta s mutation and a beta +-thalassemia in cis.
- Published
- 1987
50. Asymptomatic association of hemoglobin Dunn (alpha 6[A4]Asp----Asn) and hemoglobin O-Arab (beta 121[GH4]Glu----Lys) in a Moroccan man
- Author
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Henri Wajcman, Evelyne Dorléac, Jean Delaunay, Germaine Gombaud-Saintonge, J Godet, Henri Plauchu, Alain Francina, Faouzi Baklouti, and V. Baudin-Chich
- Subjects
Male ,Hemoglobins, Abnormal ,EcoRI ,Alpha (ethology) ,Peptide ,medicine.disease_cause ,Exon ,Gene mapping ,medicine ,Humans ,Trypsin ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Mutation ,biology ,Hemoglobin variants ,Hematology ,Middle Aged ,medicine.disease ,Molecular biology ,Peptide Fragments ,Morocco ,Hemoglobinopathy ,chemistry ,biology.protein ,Isoelectric Focusing - Abstract
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 presence of four alpha-genes. Hb O-Arab was easily recognized through its electrophoretic properties and was confirmed by the suppression of the Eco RI site located in exon 3 of the beta-gene. The percentages of the various hemoglobins showed that the doubly mutated hemoglobin Dunn/O-Arab has a normal stability and suggested that the Dunn mutation is carried by the alpha 1-gene. In cord blood [propositus's son], the output of the alpha Dunn gene was found equivalent to that existing in the adult.
- Published
- 1988
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