26 results on '"Faouzi Baklouti"'
Search Results
2. Proteasome-mediated proteolysis of SRSF5 splicing factor intriguingly co-occurs with SRSF5 mRNA upregulation during late erythroid differentiation.
- Author
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Osman Breig and Faouzi Baklouti
- Subjects
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. SMA-linked SMN mutants prevent phase separation properties and SMN interactions with FMRP family members
- Author
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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
- Subjects
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.
- Published
- 2022
4. Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop
- Author
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Alexandre Douablin, Faouzi Baklouti, Osman Breig, and Orianne Théoleyre
- Subjects
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.
- Published
- 2010
5. Cryptic splicing sites are differentially utilized in vivo
- Author
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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.
- Published
- 2008
6. Des oncogènes à l’interface entre transcription et épissage
- Author
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Faouzi Baklouti and Orianne Théoleyre
- Subjects
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
7. 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
8. 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
9. 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
10. 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
11. 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
12. 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
13. 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
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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
14. Cryptic splicing sites are differentially utilized in vivo
- Author
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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
15. [Oncogenic transcription factors as splicing regulators]
- Author
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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
16. Spi-1/PU.1 but not Fli-1 inhibits erythroid-specific alternative splicing of 4.1R pre-mRNA in murine erythroleukemia cells
- Author
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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
17. JAK2V617F mRNA metabolism in myeloproliferative neoplasm cell lines
- Author
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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
18. Multiple cis elements regulate an alternative splicing event at 4.1R pre-mRNA during erythroid differentiation
- Author
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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
19. Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA
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Jean Delaunay, Shu-Ching Huang, Edward J. Benz, Faouzi Baklouti, and Tom Vulliamy
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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.
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- 1997
20. Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation
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Faouzi Baklouti and Osman Breig
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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.
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- 2013
21. [Untitled]
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Natacha Dreumont, Harvey Louis Levy, Jacques Poudrier, Anne Bergeron, Faouzi Baklouti, and Robert M. Tanguay
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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
22. Increased oxygen affinity with normal heterotropic effects in hemoglobin Loire [α88(F9)Ala→Ser]
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Georges Teyssier, Henri Wajcman, Jean Delaunay, Jean Kister, Faouzi Baklouti, V. Baudin-Chich, Claude Poyart, and Michael C. Marden
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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.
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- 1988
23. Hemoglobin Grange-Blanche [β27(B9) Ala → Val], a new variant with normal expression and increased affinity for oxygen
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G. Richard, Jean Delaunay, C. Périer, J. Jaubert, A. Geyssant, Alain Francina, Y. Giraud, C. Brizard, and Faouzi Baklouti
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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.
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24. δ°-Thalassemia in cis of βKnossos-globin gene Normal structure and normal transient expression of the δ-globin gene
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Colette Gonnet, Jean Delaunay, Faouzi Baklouti, D. Bozon, J Godet, and R. Ouazana
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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.
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25. Association in cis of beta +-thalassemia and hemoglobin S
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Jean Delaunay, Faouzi Baklouti, Alain Francina, Georges Richard, Evelyne Dorléac, J Godet, and Daniel Rosenberg
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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.
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- 1987
26. Asymptomatic association of hemoglobin Dunn (alpha 6[A4]Asp----Asn) and hemoglobin O-Arab (beta 121[GH4]Glu----Lys) in a Moroccan man
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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
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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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