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1.
PLoS Pathog ; 16(8): e1008346, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764824

RESUMO

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Though circPSD3 has been shown to bind factor eIF4A3 that modulates the cellular nonsense-mediated decay (NMD) pathway, circPSD3 regulates RNA amplification in a pro-viral manner at a post-translational step, while eIF4A3 exhibits the anti-viral property of the NMD pathway. Findings from the global analyses of the circular RNA landscape argue that pro-, and likely, anti-viral functions are executed by circRNAs that modulate viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis.


Assuntos
Carcinoma Hepatocelular/virologia , Hepacivirus/genética , Hepatite C/complicações , Neoplasias Hepáticas/virologia , Provírus/genética , RNA Circular/genética , RNA Viral/genética , Replicação Viral , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Nat Commun ; 11(1): 4287, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855419

RESUMO

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.


Assuntos
Anormalidades Múltiplas/etiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Troca de Cromátide Irmã , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proliferação de Células , RNA Helicases DEAD-box/química , DNA Helicases/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Estabilidade Proteica , Pseudogenes , RNA Helicases/genética , RNA Helicases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Síndrome , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
Medicine (Baltimore) ; 99(27): e20806, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629665

RESUMO

RATIONALE: DICER1 syndrome is an autosomal-dominant tumor predisposition syndrome associated with numerous cancerous and noncancerous conditions. The most common sex cord-stromal tumor associated with DICER1 syndrome is Sertoli-Leydig cell tumor of the ovary (SLCT), which is extremely unusual and accounts for < 0.5% of all ovarian neoplasms. SLCT predominantly affects adolescents and young female adults. To date, there are only a few case reports of ovarian SLCT with underlying germline DICER1 mutations. The diagnosis and treatment of this rare malignancy remains challenging in the clinic mainly due to its rarity and varied presentation. PATIENT CONCERNS: A 21-year-old Chinese girl (proband) was admitted in hospital for experiencing a lower abdominal pain and irregular vaginal bleeding for half a year. She was initially diagnosed with abdominal cavity mass prior to surgical operation. The other 20-year-old patient is the younger sister of the proband, who was diagnosed with ovarian cysts and had irregular menstruation and amenorrhea for 4 months. The elder sister underwent an uncomplicated bilateral ovarian tumor resection. Given a high degree of malignancy, comprehensive staged fertility-preserving surgery, including left adnexectomy, omentectomy, pelvic, and para-aortic lymphadenectomy, was performed. Since the other patient requested to maintain her fertility, tumor resection was only conducted in the right ovary. DIAGNOSES: The elder sister was diagnosed as poorly differentiated SLCT accompanied with heterologous stage IC rhabdomyosarcoma (RMS) based on its typical pathology features and molecular characteristics from immunohistochemistry (IHC) staining. The younger sister was diagnosed as poorly differentiated SLCT. Targeted next-generation sequencing (NGS) detected DICER1 mutation in the plasma samples and postoperative tumor tissues of both patients. INTERVENTIONS: Both patients underwent surgical tumor resection, followed by combination chemotherapy with bleomycin, etoposide, and cisplatin for 4 cycles. OUTCOMES: Patients received the above clinical interventions but eventually died from disease recurrence. The elder sister died from disease relapse after one and a half years postsurgery. The younger sister had a relapse of the disease 1 year later, but she refused the comprehensive staged surgery and died from disease relapse quickly. LESSONS: Ovarian SLCT patients with DICER1 mutations and a family history have a high degree of malignancy and are associated with a poor prognosis. With ongoing research efforts on DICER1 mutations, genetic screening and counselling on a regular basis is recommended for predicting potential future cancer risk of individuals with DICER1 syndrome family history.


Assuntos
RNA Helicases DEAD-box/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ribonuclease III/genética , Tumor de Células de Sertoli-Leydig/genética , Tumor de Células de Sertoli-Leydig/patologia , Feminino , Humanos , Neoplasias Ovarianas/terapia , Tumor de Células de Sertoli-Leydig/terapia , Irmãos , Adulto Jovem
4.
PLoS Genet ; 16(7): e1008779, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730248

RESUMO

Staphylococcus aureus is an opportunistic pathogen that can grow in a wide array of conditions: on abiotic surfaces, on the skin, in the nose, in planktonic or biofilm forms and can cause many type of infections. Consequently, S. aureus must be able to adapt rapidly to these changing growth conditions, an ability largely driven at the posttranscriptional level. RNA helicases of the DEAD-box family play an important part in this process. In particular, CshA, which is part of the degradosome, is required for the rapid turnover of certain mRNAs and its deletion results in cold-sensitivity. To understand the molecular basis of this phenotype, we conducted a large genetic screen isolating 82 independent suppressors of cold growth. Full genome sequencing revealed the fatty acid synthesis pathway affected in many suppressor strains. Consistent with that result, sublethal doses of triclosan, a FASII inhibitor, can partially restore growth of a cshA mutant in the cold. Overexpression of the genes involved in branched-chain fatty acid synthesis was also able to suppress the cold-sensitivity. Using gas chromatography analysis of fatty acids, we observed an imbalance of straight and branched-chain fatty acids in the cshA mutant, compared to the wild-type. This imbalance is compensated in the suppressor strains. Thus, we reveal for the first time that the cold sensitive growth phenotype of a DEAD-box mutant can be explained, at least partially, by an improper membrane composition. The defect correlates with an accumulation of the pyruvate dehydrogenase complex mRNA, which is inefficiently degraded in absence of CshA. We propose that the resulting accumulation of acetyl-CoA fuels straight-chained fatty acid production at the expense of the branched ones. Strikingly, addition of acetate into the medium mimics the cshA deletion phenotype, resulting in cold sensitivity suppressed by the mutations found in our genetic screen or by sublethal doses of triclosan.


Assuntos
RNA Helicases DEAD-box/genética , Ácidos Graxos/metabolismo , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Ácidos Graxos/genética , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Proteínas de Membrana/genética , RNA Mensageiro/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade
5.
Mol Cell ; 79(4): 645-659.e9, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32692974

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.


Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismo
6.
Nat Commun ; 11(1): 3045, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546717

RESUMO

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.


Assuntos
Processamento Alternativo/fisiologia , Produtos do Gene tax/metabolismo , NF-kappa B/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Leucócitos Mononucleares/virologia , NF-kappa B/genética , Oncogenes , Fator de Transcrição RelA/metabolismo
7.
PLoS Genet ; 16(6): e1008808, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497036

RESUMO

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.


Assuntos
Antineoplásicos/farmacologia , Cistadenocarcinoma Seroso/genética , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/genética , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Instabilidade Cromossômica , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/secundário , RNA Helicases DEAD-box/genética , Reparo do DNA , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Estudos de Viabilidade , Feminino , Humanos , Camundongos , Camundongos Knockout , Mutação , Gradação de Tumores , Metástase Neoplásica/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/secundário , Cultura Primária de Células , Ribonuclease III/genética , Proteína Supressora de Tumor p53/genética
8.
Mol Cell ; 78(3): 493-505.e8, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353257

RESUMO

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Corpos de Inclusão Intranuclear/genética , Cromossomo Y/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células-Tronco Embrionárias/fisiologia , Endonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Corpos de Inclusão Intranuclear/metabolismo , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Enzimas Multifuncionais/genética , Família Multigênica , Estresse Oxidativo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas/genética , Fatores de Transcrição/genética , Cromossomo Y/metabolismo
9.
Neuron ; 106(3): 357-358, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32380046

RESUMO

In this issue of Neuron, Lennox et al. (2020) report the largest cohort of patients to date with DDX3X syndrome, discovering unique genotype-phenotype relationships that inform molecular pathogenesis. They then uncover unique roles of DDX3X in cortical neuron development and ribonucleoprotein granule formation.


Assuntos
RNA Helicases DEAD-box/genética , Neurogênese , Desenvolvimento Fetal , Humanos , Mutação , RNA
10.
Zhonghua Bing Li Xue Za Zhi ; 49(5): 441-447, 2020 May 08.
Artigo em Chinês | MEDLINE | ID: mdl-32392927

RESUMO

Objective: To investigate DICER1 hotspot mutations in ovarian Sertoli-Leydig cell tumor (SLCT) and its associated clinicopathological features. Methods: Forty-three SLCTs and 40 other sex cord-stromal tumors (SCSTs) diagnosed between 2010 and 2017 at Fudan University Shanghai Cancer Center were examined for somatic DICER1 hotspot mutations by Sanger sequencing. The associations between mutation status and clinicopathological features, including patient age, tumor differentiation and recurrence, were analyzed. Results: Somatic DICER1 mutations were found in 51% (22/43) of SLCTs, while none in the other 40 SCSTs. The most common mutation of DICER1 was p.D1709N in exon 24 (41%, 9/22) and the second most common mutation of DICER1 was p.E1813K in exon 25 (14%, 3/22). A novel frameshift mutation (c.5464delG, p.M1837fs*16) was identified in one SLCT with microcystic pattern. Mutations were more likely to occur in patients under forty years of age (P=0.046), whereas no significant associations were found between DICER1 mutations and clinical symptoms, morphology or tumor recurrence. Conclusions: Somatic DCIER1 hotspot mutations are specifically found in SLCT and may serve as an ancillary marker in differential diagnosis of SLCT from other SCST. The mutations occur more often in young patients (<40 years old). Additional studies are warranted to examine the associations between DICER1 mutations and clinicopathological features and prognosis of SLCT.


Assuntos
RNA Helicases DEAD-box/genética , Neoplasias Ovarianas , Ribonuclease III/genética , Tumor de Células de Sertoli-Leydig , Adulto , China , Feminino , Humanos , Mutação , Neoplasias Ovarianas/genética , Tumor de Células de Sertoli-Leydig/genética , Tumores do Estroma Gonadal e dos Cordões Sexuais
11.
RNA ; 26(9): 1160-1169, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32414857

RESUMO

DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli). We show that deaD transcription initiates 838 nt upstream of the start of the coding region. We have also found that DeaD is autoregulated through a negative feedback mechanism that operates both at the level of deaD mRNA stability and Rho-dependent transcription termination, and this regulation is dependent upon its mRNA 5' untranslated region (5' UTR). These findings suggest that DeaD might be regulating the conformation of its own mRNA through its RNA helicase activity to facilitate ribonuclease and Rho access to its 5' UTR.


Assuntos
RNA Helicases DEAD-box/genética , Escherichia coli/genética , Homeostase/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transcrição Genética/genética , Regiões 5' não Traduzidas/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Bacteriano/genética
12.
PLoS Negl Trop Dis ; 14(4): e0008105, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251473

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.


Assuntos
Expressão Gênica , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/genética , Interações Hospedeiro-Patógeno/genética , Fígado/metabolismo , RNA-Seq/métodos , 2',5'-Oligoadenilato Sintetase/genética , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Redes Reguladoras de Genes , Febre Hemorrágica da Crimeia/metabolismo , Febre Hemorrágica da Crimeia/virologia , Células Hep G2 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , RNA Mensageiro , Transdução de Sinais , Receptor Toll-Like 9 , Replicação Viral , Sequenciamento Completo do Exoma
13.
Nat Cell Biol ; 22(4): 372-379, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231306

RESUMO

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.


Assuntos
RNA Helicases DEAD-box/genética , Melanócitos/metabolismo , Crista Neural/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Receptores de Progesterona/genética , Proteínas de Peixe-Zebra/genética , Animais , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Leflunomida/farmacologia , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Crista Neural/efeitos dos fármacos , Crista Neural/crescimento & desenvolvimento , Nucleotídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Ligação Proteica , Receptores de Progesterona/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(15): 8573-8583, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32220961

RESUMO

Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an ∼50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-ß and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of ∼170- and ∼50-kDa Dicer fragments. Mass spectrometry identified the Dicer fragments and showed cleavage about 450 residues upstream from the C terminus. Also, PGE2 (prostaglandin E2) added to differentiating MM6 cells up-regulated full-length Dicer, through EP2/EP4 and cAMP. The TLR stimuli strongly induced miR-146a-5p, while PGE2 increased miR-99a-5p and miR-125a-5p, both implicated in down-regulation of TNFα. The Ser protease inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride) up-regulated full-length Dicer, both in MM6 cells and in primary human blood monocytes, indicating a specific proteolytic degradation. However, AEBSF alone did not lead to a general increase in miR expression, indicating that additional mechanisms are required to increase miRNA biosynthesis. Finally, differentiation of monocytes to macrophages with M-CSF or GM-CSF strongly up-regulated full-length Dicer. Our results suggest that differentiation regimens, both in the MM6 cell line and of peripheral blood monocytes, inhibit an apparently constitutive Dicer proteolysis, allowing for increased formation of miRNAs.


Assuntos
Diferenciação Celular , RNA Helicases DEAD-box/metabolismo , MicroRNAs/metabolismo , Monócitos/metabolismo , Prostaglandina-E Sintases/metabolismo , Proteólise , Ribonuclease III/metabolismo , Células Cultivadas , RNA Helicases DEAD-box/genética , Dinoprostona/farmacologia , Hematopoese , Humanos , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Monócitos/citologia , Monócitos/efeitos dos fármacos , Prostaglandina-E Sintases/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Ribonuclease III/genética , Zimosan/farmacologia
15.
Neuron ; 106(3): 404-420.e8, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32135084

RESUMO

De novo germline mutations in the RNA helicase DDX3X account for 1%-3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease.


Assuntos
Córtex Cerebral/metabolismo , RNA Helicases DEAD-box/genética , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/genética , Neurogênese , Animais , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/anormalidades , Córtex Cerebral/embriologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos do Neurodesenvolvimento/patologia , RNA/metabolismo
16.
Mol Cell ; 78(2): 317-328.e6, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32191872

RESUMO

MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.


Assuntos
Proteínas Argonauta/genética , Eritropoese/genética , MicroRNAs/genética , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Interferência de RNA , Ribonuclease III/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
17.
Virchows Arch ; 477(3): 375-382, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32193603

RESUMO

Congenital pulmonary airway malformation (CPAM) occurs most commonly in infants. It is divided into 5 types. The most common types 1 and 2 are cystic, type 0 presents as bronchial buds without alveolar tissue, most likely corresponding to alveolar dysgenesis, while type 3 is composed of branching bronchioles and appears as a solid lesion. A defect in the epithelial-mesenchymal crosstalk might be the underlying mechanism for all. Type 4 is a peripheral cystic lesion with a thin cyst wall covered by pneumocytes. CPAM 4 has been mixed up with pleuropulmonary blastoma (PPB) type I and some authors question its existence. We investigated five cases of CPAM type 4 for the presence or absence of rhabdomyoblasts, and for markers associated with CPAM development. In addition, all cases were evaluated for mutations within the Dicer gene and for mutations of the RAS family of oncogenes. All five cases showed smooth muscle actin and desmin-positive cells; however, only one case showed a few cells positive for MyoD. The same case showed a mutation of Dicer 1. All cases were negative for mutations of the RAS family of genes. Fibroblast growth factor 10 was similarly expressed in all cases, and thus cannot be used to differentiate CPAM4 from PPB-I. Low expression of the proliferation marker Ki67 was seen in our CPAM 4 cases and the probable PPB-I case. YingYang-1 protein seems to play an active role in the development of PPB-I. CPAM 4 can be separated from PPB-I based on the presence of rhabdomyoblasts and mutations in Dicer 1 gene. These cells might not be numerous; therefore, all available tissue has to be evaluated. As CPAM 4 morphologically looks very similar to PPB-I, it might be speculated, that there exists a potential for progression from CPAM 4 to PPB-I, by acquiring somatic mutations in Dicer 1.


Assuntos
Malformação Adenomatoide Cística Congênita do Pulmão/patologia , RNA Helicases DEAD-box/genética , Blastoma Pulmonar/etiologia , Blastoma Pulmonar/genética , Ribonuclease III/genética , Adolescente , Biomarcadores Tumorais/genética , Malformação Adenomatoide Cística Congênita do Pulmão/complicações , Malformação Adenomatoide Cística Congênita do Pulmão/diagnóstico , Diagnóstico Diferencial , Progressão da Doença , Feminino , Genes ras , Humanos , Lactente , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Adulto Jovem
18.
Rev Invest Clin ; 72(1): 19-24, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32132739

RESUMO

Background: Previous studies have shown an association between polymorphisms of the BAT1-NF-κB inhibitor-like-1 (NFKBIL1)-LTA genomic region and susceptibility to myocardial infarction and acute coronary syndrome (ACS). Objective: The objective of the study was to study the role of three polymorphisms in the BAT1, NFKBIL1, and LTA genes on the susceptibility or protection against ACS; we included a group of cases-controls from Central Mexico. Methods: The BAT1 rs2239527C/G, NFKBIL1 rs2071592T/A, and LTA rs1800683G/A polymorphisms were genotyped using a 5' TaqMan assay in a group of 625 patients with ACS and 617 healthy controls. Results: Under a recessive model, the BAT1 -23C/G (rs2239527) polymorphism showed an association with protection against ACS (odds ratio = 0.56, and p-corrected = 0.019). In contrast, the genotype and allele frequencies of the NFKBIL1 rs2071592T/A and LTA rs1800683G/A polymorphisms were similar between ACS patients and controls and no association was identified. Conclusion: Our data suggest an association between the BAT1 -23C/G polymorphism and protection against ACS in Mexican patients.


Assuntos
Síndrome Coronariana Aguda/genética , RNA Helicases DEAD-box/genética , Infarto do Miocárdio/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Linfotoxina-alfa/genética , Masculino , México , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
19.
BMC Biol ; 18(1): 32, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209106

RESUMO

BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.


Assuntos
RNA Helicases DEAD-box/genética , NF-kappa B/metabolismo , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais , Alquilação , Animais , RNA Helicases DEAD-box/metabolismo , Sondas de DNA , Tratamento Farmacológico , Humanos , Camundongos , Receptores de Reconhecimento de Padrão/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-32213379

RESUMO

Cold stress is one of the most common unfavorable environmental factors affecting the growth, development, and survival of plants. The DEAD-box RNA helicases play important roles in all types of processes of RNA metabolism. However, the function of DEAD-box RNA helicase under cold stress is poorly explored in plants, especially in rice. This study reported the identification of a novel rice thermo-sensitive chlorophyll-deficient mutant, tcd33, which displayed an albino phenotype before the four-leaf stage, then withered and eventually died at 20 °C, while wild-type plants exhibited normal green coloration at 32 °C. The tcd33 seedlings also exhibited less chlorophyll contents and severe defects of chloroplast structure under 20 °C condition. Map-based cloning and complementation experiments suggested that TCD33 encodes a chloroplast-located DEAD-box RNA helicase protein. The transcript expression level of TCD33 indicated that the genes related to chlorophyll (Chl) biosynthesis, photosynthesis, and chloroplast development in tcd33 mutants were down-regulated at 20 °C, while the down-regulated genes were nearly recovered to or slightly higher than the WT level at 32 °C. Together, our results suggest that the cold-inducible TCD33 is essential for early chloroplast development and is important for cold-responsive gene regulation and cold tolerance in rice.


Assuntos
Cloroplastos/metabolismo , Resposta ao Choque Frio/genética , RNA Helicases DEAD-box/genética , Oryza/fisiologia , Proteínas de Plantas/genética , Sequência de Aminoácidos , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plântula/metabolismo , Alinhamento de Sequência
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