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1.
Nat Commun ; 11(1): 4737, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968070

RESUMO

Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P < 0.0001; 95% prediction interval, 0.618 to 0.734). Meanwhile, an NRTI, lamivudine, improves insulin sensitivity and reduces inflammasome activation in diabetic and insulin resistance-induced human cells, as well as in mice fed with high-fat chow; mechanistically, inflammasome-activating short interspersed nuclear element (SINE) transcripts are elevated, whereas SINE-catabolizing DICER1 is reduced, in diabetic cells and mice. These data suggest the possibility of repurposing an approved class of drugs for prevention of diabetes.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Reposicionamento de Medicamentos , Inflamassomos/efeitos dos fármacos , Resistência à Insulina , Inibidores da Transcriptase Reversa/farmacologia , Adipócitos/metabolismo , Animais , Sobrevivência Celular , RNA Helicases DEAD-box/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , HIV-1/efeitos dos fármacos , Hepatite B , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/metabolismo , Ribonuclease III/metabolismo
2.
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
3.
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
4.
Gene ; 761: 145037, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777526

RESUMO

Primordial germ cells (PGCs) are singled out from somatic cells very early during embryogenesis, then they migrate towards the genital ridge and differentiate into gametes through oogenesis or spermatogenesis. Labeling PGCs with Localized RNAexpression (LRE) technique by fluorescent proteins has been widely applied among teleost species to study the germ cell development and gonad differentiation. In this study, we first cloned and characterized the 3' untranslated regions (3'UTRs) of nanos homolog 1-like (nos1l), dead end (dnd), and vasa in yellow catfish (Pelteobagrus fulvidraco), and then synthesized the GFP-nos1l/dnd/vasa 3'UTR mRNAs. Each of these three 3'UTRs could label PGCs in yellow catfish embryos, of which, vasa 3'UTR exhibited the highest labeling efficiency. To identify the differences in PGCs at embryonic stage, XX all-female and XY all-male yellow catfish embryos were produced and injected with GFP-vasa 3'UTR mRNA. We observed the PGC migration route in these two monosex embryos from 24 hpf to 7 dpf, and found there was no difference between them. Besides, the PGC number was counted at 48 hpf, and the result showed that the average PGC number in XX females (11.3) was significantly larger than that in XY males (8.1).These findings provide an insight into the development of PGCs in yellow catfish embryos and the relationship between embryonicPGCnumberand thelatergonaddifferentiation.


Assuntos
Peixes-Gato/genética , Gametogênese/genética , Células Germinativas/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Movimento Celular/genética , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/metabolismo , Masculino , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo
5.
Mol Cell ; 79(4): 629-644.e4, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32679035

RESUMO

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.


Assuntos
Ribossomos Mitocondriais/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/química , Trypanosoma brucei brucei/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Ribossômico/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Trypanosoma brucei brucei/genética
6.
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
7.
Nat Commun ; 11(1): 3181, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576832

RESUMO

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , RNA Helicases DEAD-box/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Conversão Gênica , Instabilidade Genômica , Recombinação Homóloga , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
8.
Nat Commun ; 11(1): 3122, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561742

RESUMO

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Espectrometria de Massas , Mutação , Mapeamento de Interação de Proteínas , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA-Seq , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
9.
Nature ; 583(7815): 310-313, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32494006

RESUMO

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52-7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.


Assuntos
Microscopia Crioeletrônica , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/ultraestrutura , Sequência de Bases , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Transativadores/química , Transativadores/metabolismo
10.
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
11.
Nucleic Acids Res ; 48(13): 7218-7238, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542338

RESUMO

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estruturas R-Loop , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo
12.
DNA Cell Biol ; 39(7): 1091-1095, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32397752

RESUMO

The crosstalk between cellular stress responses and innate immune signaling pathways remains poorly understood. Cells can respond to stressors by assembling stress granules that store 40S ribosomes, translation initiation factors, and mRNAs, and allow the cell to survive. Some stressors can activate the NLRP3 inflammasome, which leads to pyroptotic cell death. Stress granules and the NLRP3 inflammasome provide distinct cell fate choices to the cell. These complexes also involve distinct types of phase transitions-liquid-liquid phase separation for stress granules and prionoid phase transition for the NLRP3 inflammasome. We recently reported that DDX3X modulates this crosstalk by acting as a common essential factor for NLRP3 inflammasome activation and stress granule assembly. Here, we discuss the role of DDX3X in modulating the liquid-liquid phase separation and prionoid phase transition required for making cell fate decisions under stress conditions.


Assuntos
Morte Celular , RNA Helicases DEAD-box/metabolismo , Transdução de Sinais , Animais , Humanos , Receptor Cross-Talk
13.
Nat Commun ; 11(1): 2661, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461552

RESUMO

RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma.


Assuntos
Quadruplex G , Glioblastoma/fisiopatologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica/fisiologia , Instabilidade Genômica/fisiologia , Humanos , RNA Mensageiro/metabolismo
14.
Nucleic Acids Res ; 48(11): 6340-6352, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383752

RESUMO

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5-FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , Ciclina D1/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo
15.
Mol Cell ; 78(6): 1224-1236.e5, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32442398

RESUMO

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.


Assuntos
MicroRNAs/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Camundongos , MicroRNAs/genética , Cultura Primária de Células , RNA Nucleotidiltransferases/metabolismo , Ribonuclease III/metabolismo
16.
Nucleic Acids Res ; 48(11): 5799-5813, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32399566

RESUMO

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.


Assuntos
RNA Helicases DEAD-box/metabolismo , Processamento de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Alelos , Genes Fúngicos/genética , Genoma Fúngico/genética , Mutação , Fenótipo , Ligação Proteica , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
17.
Mol Cell Biol ; 40(13)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32312884

RESUMO

RNA helicase DHX33 was found to regulate the transcription of multiple genes involved in cancer development. But the underlying molecular mechanism remains unclear. Here, we found DHX33 associated extensively with gene promoters at CG-rich region. Its deficiency reduced the loading of active RNA polymerase II at gene promoters. Furthermore, we observed a functional interaction between DHX33, AP-2ß, and DNA demethylation protein Gadd45a (growth arrest and DNA damage inductile protein 45a) at specific gene promoters. DHX33 is required to recruit GADD45a, thereby causing local DNA demethylation through further recruiting ten-eleven-translocation (Tet) methylcytosine dioxygenase enzyme, as manifested by reduced 5-hydroxymethyl cytosine levels for a subset of genes after DHX33 deficiency. This process might involve R-loop formation in GC skew as a guidance signal at promoter sites. Our report provides for the first time, to our knowledge, original evidence that DHX33 alters epigenetic marks and regulates specific gene transcription through interaction with Gadd45a.


Assuntos
Proteínas de Ciclo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Desmetilação do DNA , Neoplasias/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Ativação Transcricional
18.
PLoS Pathog ; 16(4): e1008483, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32330200

RESUMO

Pathogenic hantaviruses, genus Orthohantaviridae, are maintained in rodent reservoirs with zoonotic transmission to humans occurring through inhalation of rodent excreta. Hantavirus disease in humans is characterized by localized vascular leakage and elevated levels of circulating proinflammatory cytokines. Despite the constant potential for deadly zoonotic transmission to humans, specific virus-host interactions of hantaviruses that lead to innate immune activation, and how these processes impart disease, remain unclear. In this study, we examined the mechanisms of viral recognition and innate immune activation of Hantaan orthohantavirus (HTNV) infection. We identified the RIG-I-like receptor (RLR) pathway as essential for innate immune activation, interferon (IFN) production, and interferon stimulated gene (ISG) expression in response to HTNV infection in human endothelial cells, and in murine cells representative of a non-reservoir host. Our results demonstrate that innate immune activation and signaling through the RLR pathway depends on viral replication wherein the host response can significantly restrict replication in target cells in a manner dependent on the type 1 interferon receptor (IFNAR). Importantly, following HTNV infection of a non-reservoir host murine model, IFNAR-deficient mice had higher viral loads, increased persistence, and greater viral dissemination to lung, spleen, and kidney compared to wild-type animals. Surprisingly, this response was MAVS independent in vivo. Innate immune profiling in these tissues demonstrates that HTNV infection triggers expression of IFN-regulated cytokines early during infection. We conclude that the RLR pathway is essential for recognition of HTNV infection to direct innate immune activation and control of viral replication in vitro, and that additional virus sensing and innate immune response pathways of IFN and cytokine regulation contribute to control of HTNV in vivo. These results reveal a critical role for innate immune regulation in driving divergent outcomes of HTNV infection, and serve to inform studies to identify therapeutic targets to alleviate human hantavirus disease.


Assuntos
Proteína DEAD-box 58/imunologia , Infecções por Hantavirus/imunologia , Hantavirus/fisiologia , Interferon Tipo I/imunologia , Replicação Viral/fisiologia , Animais , Chlorocebus aethiops , Citocinas/imunologia , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , RNA Helicases DEAD-box/metabolismo , Células Endoteliais/metabolismo , Hantavirus/imunologia , Hantavirus/metabolismo , Hantavirus/patogenicidade , Infecções por Hantavirus/metabolismo , Infecções por Hantavirus/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Interferon beta/metabolismo , Camundongos , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/imunologia , Células Vero
20.
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
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