Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
1.
Nucleic Acids Res ; 52(6): 3050-3068, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38224452

RESUMO

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Precursores de RNA/metabolismo , Fatores de Transcrição/metabolismo , DNA , Proteínas de Ligação a RNA/metabolismo
2.
Nucleic Acids Res ; 52(5): e26, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38281241

RESUMO

RNA-protein interactions determine the cellular fate of RNA and are central to regulating gene expression outcomes in health and disease. To date, no method exists that is able to identify proteins that interact with specific regions within endogenous RNAs in live cells. Here, we develop SHIFTR (Selective RNase H-mediated interactome framing for target RNA regions), an efficient and scalable approach to identify proteins bound to selected regions within endogenous RNAs using mass spectrometry. Compared to state-of-the-art techniques, SHIFTR is superior in accuracy, captures minimal background interactions and requires orders of magnitude lower input material. We establish SHIFTR workflows for targeting RNA classes of different length and abundance, including short and long non-coding RNAs, as well as mRNAs and demonstrate that SHIFTR is compatible with sequentially mapping interactomes for multiple target RNAs in a single experiment. Using SHIFTR, we comprehensively identify interactions of cis-regulatory elements located at the 5' and 3'-terminal regions of authentic SARS-CoV-2 RNAs in infected cells and accurately recover known and novel interactions linked to the function of these viral RNA elements. SHIFTR enables the systematic mapping of region-resolved RNA interactomes for any RNA in any cell type and has the potential to revolutionize our understanding of transcriptomes and their regulation.


Assuntos
Proteômica , Proteínas de Ligação a RNA , RNA , Software , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Transcriptoma , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Proteômica/métodos
3.
Cell ; 186(22): 4834-4850.e23, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37794589

RESUMO

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.


Assuntos
COVID-19 , RNA Viral , Humanos , COVID-19/metabolismo , Endonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação Viral
4.
Nat Commun ; 13(1): 1018, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35197461

RESUMO

The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remains unclear. Here, we combine a multi-omics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype associated with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies.


Assuntos
COVID-19/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Assistência Ambulatorial , Citocinas/sangue , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Interferons/imunologia , Células Matadoras Naturais/imunologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Nasofaringe/imunologia , Nasofaringe/virologia , SARS-CoV-2/fisiologia , Linfócitos T/imunologia
5.
Blood ; 139(16): 2534-2546, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35030251

RESUMO

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.


Assuntos
Anemia , Fator de Transcrição GATA1 , Diferenciação Celular/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Eritropoese/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Humanos
6.
Biospektrum (Heidelb) ; 27(4): 376-379, 2021.
Artigo em Alemão | MEDLINE | ID: mdl-34219983

RESUMO

Using RNA antisense purification and mass spectrometry, we identified more than 100 human proteins that directly and specifically bind SARS-CoV-2 RNA in infected cells. To gain insights into the functions of selected RNA interactors, we applied genetic perturbation and pharmacological inhibition experiments, and mapped the contact sites on the viral RNA. This led to the identification of host dependency factors and defense strategies, which can guide the design of novel therapeutics against SARS-CoV-2.

7.
mBio ; 12(3)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947766

RESUMO

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a ß-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication.IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.


Assuntos
Citomegalovirus/genética , Interações entre Hospedeiro e Microrganismos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Células Cultivadas , Citomegalovirus/fisiologia , Fibroblastos/virologia , Células HEK293 , Humanos , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA/farmacologia , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Replicação Viral/genética
8.
Nat Microbiol ; 6(3): 339-353, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33349665

RESUMO

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo , Autoantígenos/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Mapas de Interação de Proteínas , Proteoma , RNA Viral/genética , Ribonucleoproteínas/metabolismo , SARS-CoV-2/genética , Replicação Viral/fisiologia , Antígeno SS-B
9.
Nat Commun ; 11(1): 3214, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587247

RESUMO

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.


Assuntos
Regulação Neoplásica da Expressão Gênica , Nucleotídeos/metabolismo , RNA Longo não Codificante/genética , Ribonucleosídeo Difosfato Redutase , Proteína 1 de Ligação a Y-Box , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
10.
Nat Genet ; 52(2): 138-145, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959994

RESUMO

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and ß-thalassemia1. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development2-7. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.


Assuntos
Hemoglobinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Adulto , Animais , Sítios de Ligação , Células Cultivadas , Células Eritroides/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica , Hemoglobinas/genética , Humanos , Recém-Nascido , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
11.
Genes Dev ; 33(23-24): 1673-1687, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31699777

RESUMO

Knockout of the ubiquitously expressed miRNA-17∼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17∼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17∼92:Bim interactions to the complex miR-17∼92 knockout phenotype, we used a system of conditional mutagenesis of the nine Bim 3' UTR miR-17∼92 seed matches. Blocking miR-17∼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17∼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by Bim haploinsufficiency. Thus, the interaction of miR-17∼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.


Assuntos
Linfócitos B/citologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Sobrevivência Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Linfócitos B/patologia , Proteína 11 Semelhante a Bcl-2/genética , Técnicas de Inativação de Genes , Pulmão/embriologia , Camundongos , MicroRNAs/genética , Mutação , Estresse Fisiológico
13.
Elife ; 72018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30272558

RESUMO

Maintaining a healthy proteome involves all layers of gene expression regulation. By quantifying temporal changes of the transcriptome, translatome, proteome, and RNA-protein interactome in cervical cancer cells, we systematically characterize the molecular landscape in response to proteostatic challenges. We identify shared and specific responses to misfolded proteins and to oxidative stress, two conditions that are tightly linked. We reveal new aspects of the unfolded protein response, including many genes that escape global translation shutdown. A subset of these genes supports rerouting of energy production in the mitochondria. We also find that many genes change at multiple levels, in either the same or opposing directions, and at different time points. We highlight a variety of putative regulatory pathways, including the stress-dependent alternative splicing of aminoacyl-tRNA synthetases, and protein-RNA binding within the 3' untranslated region of molecular chaperones. These results illustrate the potential of this information-rich resource.


Assuntos
Proteostase , Estresse Fisiológico , Aminoacil-tRNA Sintetases/metabolismo , Reparo do DNA/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Essenciais , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Análise de Componente Principal , Biossíntese de Proteínas/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Proteostase/genética , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/metabolismo
14.
Nature ; 561(7721): 132-136, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30150775

RESUMO

The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen2-7. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability8, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Instabilidade Genômica , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Segregação de Cromossomos , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Espectrometria de Massas , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Processamento de RNA/metabolismo , RNA Longo não Codificante/genética , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo
16.
Cell ; 173(1): 90-103.e19, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29551269

RESUMO

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.


Assuntos
Anemia de Diamond-Blackfan/patologia , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Anemia de Diamond-Blackfan/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Mutação de Sentido Incorreto , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Am J Hematol ; 92(9): E513-E519, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28568895

RESUMO

Immunodeficient mouse models have been valuable for studies of human hematopoiesis, but high-fidelity recapitulation of erythropoiesis in most xenograft recipients remains elusive. Recently developed immunodeficient and Kit mutant mice, however, have provided a suitable background to achieve higher-level human erythropoiesis after long-term hematopoietic engraftment. While there has been some characterization of human erythropoiesis in these models, a comprehensive analysis from various human developmental stages has not yet been reported. Here, we have utilized cell surface phenotypes, morphologic analyses, and molecular studies to fully characterize human erythropoiesis from multiple developmental stages in immunodeficient and Kit mutant mouse models following long-term hematopoietic stem and progenitor cell engraftment. We show that human erythropoiesis in such models demonstrates complete maturation and enucleation, as well as developmentally appropriate globin gene expression. These results provide a framework for future studies to utilize this model system for interrogating disorders affecting human erythropoiesis and for developing improved therapeutic approaches.


Assuntos
Eritropoese , Transplante de Células-Tronco Hematopoéticas , Mutação , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-kit/genética
18.
Science ; 354(6313): 769-773, 2016 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-27708057

RESUMO

Gene expression in mammals is regulated by noncoding elements that can affect physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase of sequence in the vicinity of two essential transcription factors, MYC and GATA1, and identify nine distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the seven enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer-promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease.


Assuntos
Mapeamento Cromossômico/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas/fisiologia , Sistemas CRISPR-Cas , Proliferação de Células/genética , Doença/genética , Elementos Facilitadores Genéticos/genética , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Humanos , Células K562 , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Reação em Cadeia da Polimerase em Tempo Real
19.
Biomolecules ; 5(3): 1441-66, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26184334

RESUMO

DDX6 (p54/RCK) is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58) of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2) and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2). We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6's multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1ß, we tested for and observed a significant enrichment of sumoylation amongst DDX6's interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions-many of which are likely conserved across eukaryotes.


Assuntos
Citosol/metabolismo , RNA Helicases DEAD-box/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Células HEK293 , Resposta ao Choque Térmico , Humanos , Estresse Oxidativo , Transporte Proteico
20.
Mol Cell ; 54(4): 573-85, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24726324

RESUMO

RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs.


Assuntos
Regiões 3' não Traduzidas , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/genética , Motivos de Aminoácidos , Sítios de Ligação , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA