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1.
Blood ; 137(1): 89-102, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-32818241

RESUMO

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.


Assuntos
Diferenciação Celular/fisiologia , Células Eritroides/citologia , Eritropoese/fisiologia , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Células-Tronco Hematopoéticas , Humanos , Camundongos , Biogênese de Organelas
2.
Haematologica ; 106(3): 746-758, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32327500

RESUMO

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3'UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3'UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.


Assuntos
Anemia de Diamond-Blackfan , Anemia Macrocítica , Proteínas Ribossômicas , Anemia de Diamond-Blackfan/genética , Humanos , Proteoma/genética , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Ribossomos/genética
3.
PLoS Genet ; 8(3): e1002608, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22479203

RESUMO

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the -1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.


Assuntos
Aminoácidos/genética , Códon sem Sentido , Gentamicinas , Terminação Traducional da Cadeia Peptídica , RNA de Transferência/genética , Células Cultivadas , Códon sem Sentido/efeitos dos fármacos , Códon de Terminação/efeitos dos fármacos , Citosina , Gentamicinas/farmacologia , Humanos , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Uracila
4.
Blood ; 119(6): 1532-42, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22160620

RESUMO

Normal human erythroid cell maturation requests the transcription factor GATA-1 and a transient activation of caspase-3, with GATA-1 being protected from caspase-3-mediated cleavage by interaction with the chaperone heat shock protein 70 (Hsp70) in the nucleus. Erythroid cell dysplasia observed in early myelodysplastic syndromes (MDS) involves impairment of differentiation and excess of apoptosis with a burst of caspase activation. Analysis of gene expression in MDS erythroblasts obtained by ex vivo cultures demonstrates the down-regulation of a set of GATA-1 transcriptional target genes, including GYPA that encodes glycophorin A (GPA), and the up-regulation of members of the HSP70 family. GATA-1 protein expression is decreased in MDS erythroblasts, but restores in the presence of a pan-caspase inhibitor. Expression of a mutated GATA-1 that cannot be cleaved by caspase-3 rescues the transcription of GATA-1 targets, and the erythroid differentiation, but does not improve survival. Hsp70 fails to protect GATA-1 from caspases because the protein does not accumulate in the nucleus with active caspase-3. Expression of a nucleus-targeted mutant of Hsp70 protects GATA-1 and rescues MDS erythroid cell differentiation. Alteration of Hsp70 cytosolic-nuclear shuttling is a major feature of MDS that favors GATA-1 cleavage and differentiation impairment, but not apoptosis, in dysplastic erythroblasts.


Assuntos
Núcleo Celular/metabolismo , Eritropoese/genética , Fator de Transcrição GATA1/genética , Proteínas de Choque Térmico HSP70/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Eritroblastos/metabolismo , Células Eritroides/metabolismo , Feminino , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Immunoblotting , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937
5.
Nucleic Acids Res ; 39(8): 3350-62, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21149266

RESUMO

Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We identified a human cancer cell line containing a PTC, for which high levels of readthrough were obtained in the presence of aminoglycosides. Using these cells, we demonstrated that aminoglycoside treatment stabilized the mutant mRNA, which would otherwise have been degraded by non-sense-mediated decay, resulting in the production of a functional full-length p53 protein. Finally, we showed that aminoglycoside treatment decreased the viability of cancer cells specifically in the presence of nonsense-mutated p53 gene. These results open possibilities of developing promising treatments of cancers linked with non-sense mutations in tumor suppressor genes. They show that molecules designed to induce stop-codon readthrough can be used to inhibit tumor growth and offer a rational basis for developing new personalized strategies that could diversify the existing arsenal of cancer therapies.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Códon sem Sentido , Genes p53 , Amicacina/farmacologia , Animais , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Gentamicinas/farmacologia , Humanos , Camundongos , RNA Mensageiro/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/metabolismo
6.
Med Sci (Paris) ; 28(2): 193-9, 2012 Feb.
Artigo em Francês | MEDLINE | ID: mdl-22377308

RESUMO

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.


Assuntos
Códon sem Sentido/fisiologia , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Fatores de Terminação de Peptídeos/fisiologia , Alelos , Códon sem Sentido/genética , Doenças Genéticas Inatas/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/genética , Especificidade por Substrato/genética , Transcrição Gênica/genética , Transcrição Gênica/fisiologia
8.
PLoS One ; 8(4): e60961, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23637779

RESUMO

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-ß-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.


Assuntos
Trifosfato de Adenosina/metabolismo , Benzamidas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologia , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Humanos , Mesilato de Imatinib , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Piperidinas , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Piridinas
9.
PLoS One ; 6(8): e24125, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21909382

RESUMO

The APC tumor suppressor gene is frequently mutated in human colorectal cancer, with nonsense mutations accounting for 30% of all mutations in this gene. Reintroduction of the WT APC gene into cancer cells generally reduces tumorigenicity or induces apoptosis. In this study, we explored the possibility of using drugs to induce premature termination codon (PTC) readthrough (aminoglycosides, negamycin), as a means of reactivating endogenous APC. By quantifying the readthrough of 11 nonsense mutations in APC, we were able to identify those giving the highest levels of readthrough after treatment. For these mutations, we demonstrated that aminoglycoside or negamycin treatment led to a recovery of the biological activity of APC in cancer cell lines, and showed that the level of APC activity was proportional to the level of induced readthrough. These findings show that treatment with readthrough inducers should be considered as a potential strategy for treating cancers caused by nonsense mutations APC gene. They also provide a rational basis for identifying mutations responsive to readthrough inducers.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Códon sem Sentido/genética , Neoplasias Colorretais/genética , Genes APC , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , DNA Complementar/genética , Humanos , Dados de Sequência Molecular
10.
J Gene Med ; 10(2): 217-24, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18074402

RESUMO

BACKGROUND: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. METHODS: Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. RESULTS: We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. CONCLUSIONS: Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.


Assuntos
Códon sem Sentido/genética , Gentamicinas/farmacologia , Laminina/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/genética , Estabilidade de RNA/efeitos dos fármacos , Diamino Aminoácidos/farmacologia , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Miosinas/metabolismo , Células NIH 3T3 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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