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1.
Cell Death Dis ; 13(11): 940, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36347834

RESUMO

Triple-negative breast cancer (TNBC) is a great detriment to women's health due to the lack of effective therapeutic targets. In this study, we employed an integrated genetic screen to identify a pivotal oncogenic factor, heterogeneous nuclear ribonucleoprotein U (HNRNPU), which is required for the progression of TNBC. We elucidated the pro-oncogenic role of HNRNPU, which can induce the proliferation and migration of TNBC cells via its association with DEAD box helicase 5 (DDX5) protein. Elevated levels of the HNRNPU-DDX5 complex prohibited the intron retention of minichromosome maintenance protein 10 (MCM10) pre-mRNA, decreased nonsense-mediated mRNA decay, and activated Wnt/ß-catenin signalling; on the other hand, HNRNPU-DDX5 is located in the transcriptional start sites (TSS) of LIM domain only protein 4 (LMO4) and its upregulation promoted the transcription of LMO4, consequently activating PI3K-Akt-mTOR signalling. Our data highlight the synergetic effects of HNRNPU in RNA transcription and splicing in regulating cancer progression and suggest that HNRNPU may act as a potential molecular target in the treatment of TNBC.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , RNA/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Via de Sinalização Wnt , Carcinogênese , Proliferação de Células/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas com Domínio LIM/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo
2.
Nat Commun ; 13(1): 7096, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402769

RESUMO

RIG-I/DDX58 plays a key role in host innate immunity. However, its therapeutic potential for inflammation-related cancers remains to be explored. Here we identify frameshift germline mutations of RIG-I occurring in patients with colon cancer. Accordingly, Rig-ifs/fs mice bearing a frameshift mutant Rig-i exhibit increased susceptibility to colitis-related colon cancer as well as enhanced inflammatory response to chemical, virus or bacteria. In addition to interruption of Rig-i mRNA translation, the Rig-i mutation changes the secondary structure of Rig-i pre-mRNA and impairs its association with DHX9, consequently inducing a circular RNA generation from Rig-i transcript, thereby, designated as circRIG-I. CircRIG-I is frequently upregulated in colon cancers and its upregulation predicts poor outcome of colon cancer. Mechanistically, circRIG-I interacts with DDX3X, which in turn stimulates MAVS/TRAF5/TBK1 signaling cascade, eventually activating IRF3-mediated type I IFN transcription and aggravating inflammatory damage. Reciprocally, all-trans retinoic acid acts as a DHX9 agonist, ameliorates immunopathology through suppression of circRIG-I biogenesis. Collectively, our results provide insight into mutant RIG-I action and propose a potential strategy for the treatment of colon cancer.


Assuntos
Neoplasias do Colo , RNA Helicases DEAD-box , Camundongos , Animais , RNA Helicases DEAD-box/metabolismo , Transdução de Sinais , Imunidade Inata , Inflamação/genética , Neoplasias do Colo/genética
3.
Cell Death Dis ; 13(11): 926, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36335087

RESUMO

As the most important RNA epigenetic regulation in eukaryotic cells, N6-metheyladenosine (m6A) modification has been demonstrated to play significant roles in cancer progression. However, this modification in long intergenic non-coding RNAs (lincRNAs) and the corresponding functions remain elusive. Here, we showed a lincRNA LINC02551 was downregulated by AlkB Homolog 5 (ALKBH5) overexpression in a m6A-dependent manner in hepatocellular carcinoma (HCC). Functionally, LINC02551 was required for the growth and metastasis of HCC. Mechanistically, LINC02551, a bona fide m6A target of ALKBH5, acted as a molecular adaptor that blocked the combination between DDX24 and a E3 ligase TRIM27 to decrease the ubiquitination and subsequent degradation of DDX24, ultimately facilitating HCC growth and metastasis. Thus, ALKBH5-mediated LINC02551 m6A methylation was required for HCC growth and metastasis.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Epigênese Genética , RNA Helicases DEAD-box/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(48): e2203567119, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36409901

RESUMO

The DEAH/RHA helicase Prp43 remodels protein-RNA complexes during pre-messenger RNA (mRNA) splicing and ribosome biogenesis. The helicase activity and ATP turnover are intrinsically low and become activated by G-patch (gp) factors in the specific cellular context. The gp motif connects the helicase core to the flexible C-terminal domains, but it is unclear how this affects RecA domain movement during catalysis and the unwinding of RNA substrates. We developed single-molecule Förster Resonance Energy Transfer (smFRET) reporters to study RecA domain movements within Prp43 in real time. Without Pfa1(gp), the domains approach each other adopting predominantly a closed conformation. The addition of Pfa1(gp) induces an open state, which becomes even more prevalent during interaction with RNA. In the open state, Prp43 has reduced contacts with bound nucleotide and shows rapid adenosine diphosphate (ADP) release accelerating the transition from the weak (ADP) to the strong (apo) RNA binding state. Using smFRET labels on the RNA to probe substrate binding and unwinding, we demonstrate that Pfa1(gp) enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states instead of dissociating from the RNA. ATP binding to the apo-enzyme induces the translocation along the RNA, generating the unwinding force required to melt proximal RNA structures. During ATP turnover, Pfa1(gp) stimulates alternating of the RecA domains between open and closed states. Consequently, the translocation becomes faster than dissociation from the substrate in the ADP state, allowing processive movement along the RNA. We provide a mechanistic model of DEAH/RHA helicase motility and reveal the principles of Prp43 regulation by G-patch proteins.


Assuntos
RNA Helicases DEAD-box , DNA Helicases , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismo
5.
Cells ; 11(21)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36359746

RESUMO

INTRODUCTION: Several environmental stimuli may influence lupus, particularly viral infections. In this study, we used an imiquimod-induced lupus mouse model focused on the TLR7 pathway and proteomics analysis to determine the specific pathway related to viral infection and the related protein expressions in splenic B cells to obtain insight into B-cell responses to viral infection in the lupus model. MATERIALS AND METHODS: We treated FVB/N wild-type mice with imiquimod for 8 weeks to induce lupus symptoms and signs, retrieved splenocytes, selected B cells, and conducted the proteomic analysis. The B cells were co-cultured with CD40L+ feeder cells for another week before performing Western blot analysis. Panther pathway analysis was used to disclose the pathways activated and the protein-protein interactome was analyzed by the STRING database in this lupus murine model. RESULTS: The lupus model was well established and well demonstrated with serology evidence and pathology proof of lupus-mimicking organ damage. Proteomics data of splenic B cells revealed that the most important activated pathways (fold enrichment > 100) demonstrated positive regulation of the MDA5 signaling pathway, negative regulation of IP-10 production, negative regulation of chemokine (C-X-C motif) ligand 2 production, and positive regulation of the RIG-I signaling pathway. A unique protein-protein interactome containing 10 genes was discovered, within which ISG15, IFIH1, IFIT1, DDX60, and DHX58 were demonstrated to be downstream effectors of MDA5 signaling. Finally, we found B-cell intracellular cytosolic proteins via Western blot experiment and continued to observe MDA5-related pathway activation. CONCLUSION: In this experiment, we confirmed that the B cells in the lupus murine model focusing on the TLR7 pathway were activated through the MDA5 signaling pathway, an important RNA sensor implicated in the detection of viral infections and autoimmunity. The MDA5 agonist/antagonist RNAs and the detailed molecular interactions within B cells are worthy of further investigation for lupus therapy.


Assuntos
Helicase IFIH1 Induzida por Interferon , Viroses , Animais , Camundongos , RNA Helicases DEAD-box/metabolismo , Modelos Animais de Doenças , Imiquimode/farmacologia , Proteômica , Transdução de Sinais , Receptor 7 Toll-Like , Viroses/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Lúpus Eritematoso Sistêmico/induzido quimicamente
6.
Viruses ; 14(10)2022 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-36298827

RESUMO

Host-virus protein interactions are critical for intracellular viral propagation. Understanding the interactions between cellular and viral proteins may help us develop new antiviral strategies. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe damage to the global swine industry. Here, we employed co-immunoprecipitation and liquid chromatography-mass spectrometry to characterize 426 unique PEDV nucleocapsid (N) protein-binding proteins in infected Vero cells. A protein-protein interaction network (PPI) was created, and gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses revealed that the PEDV N-bound proteins belong to different cellular pathways, such as nucleic acid binding, ribonucleoprotein complex binding, RNA methyltransferase, and polymerase activities. Interactions of the PEDV N protein with 11 putative proteins: tripartite motif containing 21, DEAD-box RNA helicase 24, G3BP stress granule assembly factor 1, heat shock protein family A member 8, heat shock protein 90 alpha family class B member 1, YTH domain containing 1, nucleolin, Y-box binding protein 1, vimentin, heterogeneous nuclear ribonucleoprotein A2/B1, and karyopherin subunit alpha 1, were further confirmed by in vitro co-immunoprecipitation assay. In summary, studying an interaction network can facilitate the identification of antiviral therapeutic strategies and novel targets for PEDV infection.


Assuntos
Infecções por Coronavirus , Ácidos Nucleicos , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Chlorocebus aethiops , Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vimentina/metabolismo , Células Vero , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/metabolismo , Infecções por Coronavirus/metabolismo , Antivirais/metabolismo , RNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Metiltransferases/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , RNA Helicases DEAD-box/metabolismo , Ribonucleoproteínas/metabolismo , Carioferinas/metabolismo , Ácidos Nucleicos/metabolismo
7.
Viruses ; 14(10)2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36298646

RESUMO

In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.


Assuntos
COVID-19 , Interferons , Humanos , Interferons/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , SARS-CoV-2/genética , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Proteínas de Ligação a RNA , RNA Mensageiro/genética , Antivirais
8.
Sci Rep ; 12(1): 17259, 2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241908

RESUMO

VASA, also known as DDX4, is a member of the DEAD-box proteins and an RNA binding protein with an ATP-dependent RNA helicase. The VASA gene expression, which is required for human germ cell development, may lead to infertility. Immunocytochemistry and immunohistochemistry were used to examine the expression of VASA protein in the human testis sections of azoospermic patients, in-vitro and in-silico models. Some studies of fertile humans showed VASA expression in the basal and adluminal compartments of seminiferous tubules. Our Immunocytochemistry and immunohistochemistry in infertile humans showed expression of VASA in the luminal compartments of the seminiferous tubule. The immunohistochemical analysis of three human cases with different levels of non-obstructive azoospermia revealed a higher expression of VASA-positive cells. For this purpose, Enrichr and Shiny Gene Ontology databases were used for pathway enrichment analysis and gene ontology. STRING and Cytoscape online evaluation were applied to predict proteins' functional and molecular interactions and performed to recognize the master genes, respectively. According to the obtained results, the main molecular functions of the up-regulated and downregulated genes include the meiotic cell cycle, RNA binding, and differentiation. STRING and Cytoscape analyses presented seven genes, i.e., DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3, as the hub genes involved in infertility with VASA co-function and protein-protein interaction. Our findings suggest that VASA and its interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.


Assuntos
Azoospermia , Humanos , Masculino , Trifosfato de Adenosina/metabolismo , Azoospermia/genética , Azoospermia/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Expressão Gênica , Imuno-Histoquímica , Antígenos de Histocompatibilidade Menor/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Testículo/metabolismo
9.
Biomed Res Int ; 2022: 8610467, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36246972

RESUMO

Coxsackievirus B (CVB) 3C protease (3Cpro) plays a specific cleavage role on AU-rich binding factor (AUF1, also called hnRNP D), which consequently disputes the regulation of AUF1 on downstream molecules. In our study, the iTRAQ approach was first used to quantify the differentially expressed cellular proteins in AUF1-overexpressing HeLa cells, which provides straightforward insight into the role of AUF1 during viral infection. A total of 1,290 differentially expressed proteins (DEPs), including 882 upregulated and 408 downregulated proteins, were identified. The DEPs are involved in a variety of cellular processes via GO terms, protein-protein interactions, and a series of further bioinformatics analyses. Among the DEPs, some demonstrated important roles in cellular metabolism. In particular, DDX5 was further verified to be negatively regulated by AUF1 and increased in CVB-infected cells, which in turn promoted CVB replication. These findings provide potential novel ideas for exploring new antiviral therapy targets.


Assuntos
RNA Helicases DEAD-box , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Proteômica , Antivirais , RNA Helicases DEAD-box/metabolismo , Enterovirus Humano B/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Replicação Viral
10.
Front Immunol ; 13: 1010635, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36248895

RESUMO

Viral hemorrhagic fever (VHF) is a term referring to a group of life-threatening infections caused by several virus families (Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae). Depending on the virus, the infection can be mild and can be also characterized by an acute course with fever accompanied by hypervolemia and coagulopathy, resulting in bleeding and shock. It has been suggested that the course of the disease is strongly influenced by the activation of signaling pathways leading to RIG-I-like receptor-dependent interferon production. RIG-I-like receptors (RLRs) are one of two major receptor families that detect viral nucleic acid. RLR receptor activation is influenced by a number of factors that may have a key role in the differences that occur during the antiviral immune response in VHF. In the present study, we collected data on RLR receptors in viral hemorrhagic fevers and described factors that may influence the activation of the antiviral response. RLR receptors seem to be a good target for VHF research, which may contribute to better therapeutic and diagnostic strategies. However, due to the difficulty of conducting such studies in humans, we suggest using Lagovirus europaeus as an animal model for VHF.


Assuntos
Arenaviridae , RNA Helicases DEAD-box/metabolismo , Febres Hemorrágicas Virais , Ácidos Nucleicos , Animais , Antivirais , Humanos , Interferons
11.
Biochem Biophys Res Commun ; 634: 138-144, 2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36242920

RESUMO

In recent years, abdominal aortic aneurysm (AAA) lesions have become one of the important diseases that threaten public health. Related studies have confirmed that the occurrence of abdominal aortic aneurysms is related to inflammatory stress, cell apoptosis, and elastic fiber degradation. DDX3x is thought to interact with inflammasomes such as NLRP3 to aggravate the process of the inflammatory response, but its role in the occurrence of AAA remains unclear. Since DDX3x is indispensable in animal embryonic growth, we used an adeno-associated virus to construct gene-overexpressing mice to induce aneurysm development through AngII infusion. The results indicated that the incidence of aneurysms, inflammatory cell infiltration, vascular smooth muscle cell transformation, and oxidative stress levels were significantly increased under the condition of DDX3x overexpression. At the signaling level, activation of the AKT pathway exacerbates aneurysm formation. Taken together, we believe that DDX3x plays a key role in the development of aneurysms and may be a new target for the treatment of aneurysm progression.


Assuntos
Aneurisma da Aorta Abdominal , Camundongos , Animais , Aneurisma da Aorta Abdominal/patologia , Camundongos Knockout para ApoE , Aorta Abdominal/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos Knockout , Angiotensina II/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo
12.
Mol Cell ; 82(21): 4131-4144.e6, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36272408

RESUMO

RIG-I is an essential innate immune receptor for detecting and responding to infection by RNA viruses. RIG-I specifically recognizes the unique molecular features of viral RNA molecules and selectively distinguishes them from closely related RNAs abundant in host cells. The physical basis for this exquisite selectivity is revealed through a series of high-resolution cryo-EM structures of RIG-I in complex with host and viral RNA ligands. These studies demonstrate that RIG-I actively samples double-stranded RNAs in the cytoplasm and distinguishes them by adopting two different types of protein folds. Upon binding viral RNA, RIG-I adopts a high-affinity conformation that is conducive to signaling, while host RNA induces an autoinhibited conformation that stimulates RNA release. By coupling protein folding with RNA binding selectivity, RIG-I distinguishes RNA molecules that differ by as little as one phosphate group, thereby explaining the molecular basis for selective antiviral sensing and the induction of autoimmunity upon RIG-I dysregulation.


Assuntos
RNA Helicases DEAD-box , RNA Viral , RNA Viral/metabolismo , Ligantes , RNA Helicases DEAD-box/metabolismo , Imunidade Inata , Proteína DEAD-box 58/metabolismo , RNA de Cadeia Dupla , Proteínas de Transporte/metabolismo
13.
J Transl Med ; 20(1): 491, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36303180

RESUMO

BACKGROUND: Exosomes are deemed to be an important tool of intercellular communicators in cancer cells. Our study investigated the role of PRR34 long non-coding RNA antisense RNA 1 (PRR34-AS1) in regulating exosome secretion in hepatocellular carcinoma (HCC) cells. METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) analyzed the expression of PRR34-AS1. We assessed the function of PRR34-AS1 on the biological changes of THLE-3 cells and HCC cells. The downstream interaction between RNAS was assessed by mechanistic experiments. RESULTS: PRR34-AS1 expression was upregulated in HCC cells in comparison to THLE-3 cells. PRR34-AS1 depletion repressed HCC cell proliferation, migration and invasion as well as EMT phenotype, while PRR34-AS1 up-regulation accelerated the malignant phenotypes of THLE-3 cells. PRR34-AS1 recruited DDX3X to stabilize the mRNA level of exosomal protein Rab27a. Moreover, PRR34-AS1 facilitated the malignant phenotypes of THLE-3 cells by elevating Rab27a expression to promote the exosome secretion of VEGF and TGF-ß in HCC cells. CONCLUSIONS: The current study revealed a novel function of PRR34-AS1 in accelerating exosome secretion in HCC cells and offered an insight into lncRNA function in the regulation of tumor cell biology.


Assuntos
Carcinoma Hepatocelular , Exossomos , Neoplasias Hepáticas , MicroRNAs , RNA Longo não Codificante , Humanos , Carcinoma Hepatocelular/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias Hepáticas/patologia , Exossomos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , RNA Mensageiro/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , Linhagem Celular Tumoral , Movimento Celular/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo
14.
Nucleic Acids Res ; 50(18): 10487-10502, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36200807

RESUMO

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that have low levels of pre-existing hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.


Assuntos
RNA Helicases DEAD-box/metabolismo , Reparo do DNA , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Células HeLa , Humanos , Proteômica , Ubiquitinas/genética
15.
Biochem Biophys Res Commun ; 634: 182-188, 2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36244117

RESUMO

G-quadruplexes (G4s) are important in regulating DNA replication, repair and RNA transcription through interactions with specialized proteins. Dbp2 has been identified as a G4 DNA binding protein from Saccharomyces cerevisiae cell lysates. The majority of G4 motifs in Saccharomyces cerevisiae display 5-50 nt loops, only a few have 1-2 nt loops. Human DDX5 could unfold MycG4 DNA, whether Dbp2 also participates in remodeling G4 motifs with short loops in Saccharomyces cerevisiae remains elusive. Here we find that Dbp2 prefers G-rich substrates and binds MycG4 with a high affinity. Dbp2 possesses a dual function for different conformations of MycG4, destabilizing the folded MycG4 and inducing further folding of the unfolded MycG4. Similarly, DDX5 can unfold MycG4, but it exhibits a weaker MycG4 folding-promoting activity relative to Dbp2. Furthermore, Dbp2 facilitates DNA annealing activity in the absence of ATP, suggesting that Dbp2 can work on DNA substrates and possibly participate in DNA metabolism. Our results demonstrate that Dbp2 plays an important role in regulating the folding and unfolding activities of MycG4.


Assuntos
Quadruplex G , Proteínas de Saccharomyces cerevisiae , Humanos , RNA Helicases DEAD-box/metabolismo , DNA/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Viruses ; 14(10)2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36298642

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi's sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.


Assuntos
Herpesvirus Humano 8 , Interferon Tipo I , Sarcoma de Kaposi , Humanos , Antivirais/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Interferon Tipo I/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Viral/genética , Latência Viral/genética , Replicação Viral/genética
17.
Nat Commun ; 13(1): 5881, 2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36202822

RESUMO

The changes occurring in mRNA organization during nucleo-cytoplasmic transport and export, are not well understood. Moreover, directionality of mRNA passage through the nuclear pore complex (NPC) has not been examined within individual NPCs. Here we find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after transcription and at the nuclear periphery. Compaction levels of nuclear transcripts can be modulated by varying levels of SR proteins and by changing genome organization. Nuclear mRNPs are mostly rod-shaped with distant 5'/3'-ends, although for some, the ends are in proximity. The latter is more abundant in the cytoplasm and can be modified by translation inhibition. mRNAs and lncRNAs exiting the NPC exhibit predominant 5'-first export. In some cases, several adjacent NPCs are engaged in export of the same mRNA suggesting 'gene gating'. Altogether, we show that the mRNP is a flexible structure during travels, with 5'-directionality during export.


Assuntos
Poro Nuclear , RNA Longo não Codificante , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Dev Biol ; 492: 200-211, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36273621

RESUMO

Germ granules harbor processes that maintain germline integrity and germline stem cell capacity. Depleting core germ granule components in C. elegans leads to the reprogramming of germ cells, causing them to express markers of somatic differentiation in day-two adults. Somatic reprogramming is associated with complete sterility at this stage. The resulting germ cell atrophy and other pleiotropic defects complicate our understanding of the initiation of reprogramming and how processes within germ granules safeguard the totipotency and immortal potential of germline stem cells. To better understand the initial events of somatic reprogramming, we examined total mRNA (transcriptome) and polysome-associated mRNA (translatome) changes in a precision full-length deletion of glh-1, which encodes a homolog of the germline-specific Vasa/DDX4 DEAD-box RNA helicase. Fertile animals at a permissive temperature were analyzed as young adults, a stage that precedes by 24 â€‹h the previously determined onset of somatic reporter-gene expression in the germline. Two significant changes are observed at this early stage. First, the majority of neuropeptide-encoding transcripts increase in both the total and polysomal mRNA fractions, suggesting that GLH-1 or its effectors suppress this expression. Second, there is a significant decrease in Major Sperm Protein (MSP)-domain mRNAs when glh-1 is deleted. We find that the presence of GLH-1 helps repress spermatogenic expression during oogenesis, but boosts MSP expression to drive spermiogenesis and sperm motility. These insights define an early role for GLH-1 in repressing somatic reprogramming to maintain germline integrity.


Assuntos
Proteínas de Caenorhabditis elegans , Neuropeptídeos , Animais , Masculino , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Motilidade Espermática , Sêmen/metabolismo , Células Germinativas/metabolismo , Espermatogênese/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
19.
Cell Rep ; 40(10): 111265, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36070689

RESUMO

Germline Argonautes direct transcriptome surveillance within perinuclear membraneless organelles called nuage. In C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in and promote the formation of nuage. Previous studies have implicated GLH proteins in inherited silencing, but direct roles in small-RNA production, Argonaute binding, or mRNA targeting have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute target mRNAs, and promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates direct binding to the Argonaute WAGO-1, which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , RNA Helicases DEAD-box/metabolismo , Células Germinativas/metabolismo , RNA Mensageiro/metabolismo
20.
J Virol ; 96(18): e0115422, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36073922

RESUMO

Long noncoding RNAs (lncRNAs) have increasingly been recognized as being integral to cellular processes, including the antiviral immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is costly to the global swine industry. To identify PRRSV-related lncRNAs, we performed RNA deep sequencing and compared the profiles of lncRNAs in PRRSV-infected and uninfected Marc-145 cells. We identified a novel lncRNA called MAHAT (maintaining cell morphology-associated and highly conserved antiviral transcript; LTCON_00080558) that inhibits PRRSV replication. MAHAT binds and negatively regulates ZNF34 expression by recruiting and binding DDX6, an RNA helicase forming a complex with ZNF34. Inhibition of ZNF34 expression results in increased type I interferon expression and decreased PRRSV replication. This finding reveals a novel mechanism by which PRRSV evades the host antiviral innate immune response by downregulating the MAHAT-DDX6-ZNF34 pathway. MAHAT could be a host factor target for antiviral therapies against PRRSV infection. IMPORTANCE Long noncoding RNAs (lncRNAs) play important roles in viral infection by regulating the transcription and expression of host genes, and interferon signaling pathways. Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses in the swine industry worldwide, but the mechanisms of its pathogenesis and immunology are not fully understood. Here, a new lncRNA, designated MAHAT, was identified as a regulator of host innate immune responses. MAHAT negatively regulates the expression of its target gene, ZNF34, by recruiting and binding DDX6, an RNA helicase, forming a complex with ZNF34. Inhibition of ZNF34 expression increases type I interferon expression and decreases PRRSV replication. This finding suggests that MAHAT has potential as a new target for developing antiviral drugs against PRRSV infection.


Assuntos
Imunidade Inata , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , RNA Longo não Codificante , Replicação Viral , Animais , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Imunidade Inata/genética , Interferon Tipo I/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Suínos , Fatores Genéricos de Transcrição/metabolismo , Replicação Viral/genética
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