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1.
J Biol Chem ; 300(3): 105716, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38311174

RESUMO

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.


Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Proteína FUS de Ligação a RNA , RNA , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo
2.
Cell Rep ; 42(10): 113199, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37804508

RESUMO

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.


Assuntos
Dano ao DNA , Poli Adenosina Difosfato Ribose , Poli(ADP-Ribose) Polimerases , Proteína FUS de Ligação a RNA , Humanos , Reparo do DNA , Células HeLa , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Motivo de Reconhecimento de RNA , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo
3.
Elife ; 122023 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-36651723

RESUMO

RNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors.


Assuntos
Neoplasias , RNA , Humanos , Simulação de Dinâmica Molecular , Descoberta de Drogas , RNA Mensageiro/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
4.
Cells ; 11(23)2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36497190

RESUMO

DNA damage causes PARP1 activation in the nucleus to set up the machinery responsible for the DNA damage response. Here, we report that, in contrast to cytoplasmic PARPs, the synthesis of poly(ADP-ribose) by PARP1 opposes the formation of cytoplasmic mRNA-rich granules after arsenite exposure by reducing polysome dissociation. However, when mRNA-rich granules are pre-formed, whether in the cytoplasm or nucleus, PARP1 activation positively regulates their assembly, though without additional recruitment of poly(ADP-ribose) in stress granules. In addition, PARP1 promotes the formation of TDP-43- and FUS-rich granules in the cytoplasm, two RNA-binding proteins which form neuronal cytoplasmic inclusions observed in certain neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Together, the results therefore reveal a dual role of PARP1 activation which, on the one hand, prevents the early stage of stress granule assembly and, on the other hand, enables the persistence of cytoplasmic mRNA-rich granules in cells which may be detrimental in aging neurons.


Assuntos
Proteína FUS de Ligação a RNA , Grânulos de Estresse , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Estresse Oxidativo , Dano ao DNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Nucleic Acids Res ; 49(17): 10061-10081, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34469566

RESUMO

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.


Assuntos
Sequências Repetidas Invertidas/genética , Iniciação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/genética , Grânulos de Estresse/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Trifosfato de Adenosina/metabolismo , Arsenitos/toxicidade , Pareamento de Bases/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Ribossomos/metabolismo
6.
Commun Biol ; 4(1): 359, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742080

RESUMO

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.


Assuntos
Grânulos Citoplasmáticos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Sítios de Ligação , Proliferação de Células , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Feminino , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteína 1 de Ligação a Y-Box/genética
7.
Cell Rep ; 27(6): 1809-1821.e5, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067465

RESUMO

PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.


Assuntos
Dano ao DNA , Glicosídeo Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Compartimento Celular , Ativação Enzimática , Feminino , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidade , Modelos Biológicos , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Domínios Proteicos , Proteína FUS de Ligação a RNA/química
8.
Cells ; 9(1)2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31906126

RESUMO

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.


Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Serina/metabolismo , Transcrição Gênica , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Hibridização In Situ , Camundongos , Fosforilação , RNA Polimerase II/metabolismo , RNA Mensageiro/genética
9.
ACS Nano ; 11(7): 7189-7200, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28657719

RESUMO

TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach via atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Microscopia de Força Atômica/métodos , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo
10.
Mol Biol Cell ; 24(10): 1529-43, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23515223

RESUMO

In the organism, quiescent epithelial cells have the potential to resume cycling as a result of various stimuli, including wound healing or oxidative stress. Because quiescent cells have a low polyamine level, resuming their growth requires an increase of their intracellular polyamine levels via de novo polyamine synthesis or their uptake from plasma. Another alternative, explored here, is an intercellular exchange with polyamine-rich cycling cells via gap junctions. We show that polyamines promote gap junction communication between proliferating cells by promoting dynamical microtubule plus ends at the cell periphery and thus allow polyamine exchange between cells. In this way, cycling cells favor regrowth in adjacent cells deprived of polyamines. In addition, intercellular interactions mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration.


Assuntos
Proliferação de Células , Células Epiteliais/fisiologia , Junções Comunicantes/metabolismo , Estresse Oxidativo , Putrescina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Transporte Biológico , Comunicação Celular , Linhagem Celular , Movimento Celular , Técnicas de Cocultura , Cicloexilaminas/farmacologia , Eflornitina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Microtúbulos/metabolismo , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Poliaminas/metabolismo , Ratos , Espermina Sintase/antagonistas & inibidores , Fibras de Estresse/metabolismo
11.
Am J Physiol Cell Physiol ; 301(3): C705-16, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21677260

RESUMO

Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.


Assuntos
Adaptação Fisiológica/fisiologia , Células Epiteliais/fisiologia , Junções Comunicantes/fisiologia , Rim/fisiologia , Estresse Fisiológico/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Animais , Betaína/metabolismo , Betaína/farmacologia , Comunicação Celular/fisiologia , Contagem de Células , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/patologia , Ácido Glicirretínico/farmacologia , Células HeLa , Humanos , Soluções Hipertônicas , Inositol/metabolismo , Inositol/farmacologia , Rim/citologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Nocodazol/farmacologia , Ácidos Oleicos/farmacologia , RNA Interferente Pequeno/genética , Ratos , Solução Salina Hipertônica , Fibras de Estresse/metabolismo , Fibras de Estresse/patologia , Estresse Fisiológico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
12.
Stem Cells ; 24(12): 2723-32, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16888281

RESUMO

Bone marrow (BM) transplantation was performed on a muscular mouse model of spinal muscular atrophy that had been created by mutating the survival of motor neuron gene (Smn) in myofibers only. This model is characterized by a severe myopathy and progressive loss of muscle fibers leading to paralysis. Transplantation of wild-type BM cells following irradiation at a low dose (6 Gy) improved motor capacity (+85%). This correlated with a normalization of myofiber number associated with a higher number of regenerating myofibers (1.6-fold increase) and an activation of CD34 and Pax7 satellite cells. However, BM cells had a very limited capacity to replace or fuse to mutant myofibers (2%). These data suggest that BM transplantation was able to attenuate the myopathic phenotype through an improvement of skeletal muscle regeneration of recipient mutant mice, a process likely mediated by a biological activity of BM-derived cells. This hypothesis was further supported by the capacity of muscle protein extracts from transplanted mutant mice to promote myoblast proliferation in vitro (1.6-fold increase). In addition, a tremendous upregulation of hepatocyte growth factor (HGF), which activates quiescent satellite cells, was found in skeletal muscle of transplanted mutants compared with nontransplanted mutants. Eventually, thanks to the Cre-loxP system, we show that BM-derived muscle cells were strong candidates harboring this biological activity. Taken together, our data suggest that a biological activity is likely involved in muscle regeneration improvement mediated by BM transplantation. HGF may represent an attractive paracrine mechanism to support this activity.


Assuntos
Transplante de Medula Óssea/métodos , Atrofia Muscular Espinal/patologia , Doenças Musculares/patologia , Distrofia Muscular Animal/patologia , Fenótipo , Animais , Antígenos CD34/imunologia , Células da Medula Óssea/citologia , Proliferação de Células , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento de Hepatócito/genética , Camundongos , Camundongos Mutantes , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Fator de Transcrição PAX7/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Regeneração , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/patologia , Fator A de Crescimento do Endotélio Vascular/genética
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