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1.
Rinsho Ketsueki ; 60(7): 800-809, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31391370

RESUMO

In myeloid neoplasms, deletions of the long arm of chromosome 5 del(5q) and 7 (-7/del(7q) ) are common karyotypic abnormalities. The concurrence of del(5q) and -7/del(7q) accounts for poor prognosis in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Comprehensive analysis of copy number abnormalities and genetic mutations related to del(5q) and -7/del(7q) revealed previously cryptic pathophysiology, leading to frequent hemizygous/homozygous mutations and haploinsufficiency. In addition, detailed somatic mutations on chr5q were detected using whole-exome sequencing. CSNK1A1 and G3BP1 are located within the common deleted regions (CDRs) (5q31.1-5q33.1), and another driver gene DDX41 is present in the more telomeric region (5q35.3). All the genes mentioned above exhibited haploinsufficiency because of deletions, and low expression of G3BP1 and DDX41 correlated with poor survival. The related mutational events outside of chr5q, TP53 mutation is most frequently observed in del(5q) cases. Regarding -7/del(7q), 3 CDRs were located in 7q22, 7q34, and 7q35-36. Somatic mutations of the corresponding genes to each CDR (CUX1: 7q22, LUC7L2: 7q34, EZH2: 7q35-36) were identified, indicating that the loss of function or haploinsufficiency might result in the downstream pathological consequences. These recent findings have remarkably offered insights into genetic and clinical consequences in MDS/AML cases with del(5q) and -7/del(7q).


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 7/genética , Leucemia Mieloide Aguda/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética
2.
J Agric Food Chem ; 67(32): 8884-8895, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31345029

RESUMO

Leucine is an essential amino acid in the milk production of bovine mammary glands, but the regulatory roles and molecular mechanisms of leucine are still not known well. This study investigated the roles of leucine on milk synthesis and explored the corresponding mechanism in bovine mammary epithelial cells (BMECs). Leucine (0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM) was added to BMECs that were cultured in FBS-free OPTI-MEM medium. Leucine significantly promoted milk protein and milk fat synthesis and also increased phosphorylation of mTOR signaling protein and the protein expression levels of SREBP-1c, with the most significant effects at 0.75 mM concentration. Leucine increased the expression and nuclear localization of DDX59, and loss and gain of gene function experiments further reveal that DDX59 mediates the stimulation of leucine on the mRNA expression variation of mTOR and SREBP-1c genes. PI3K inhibition experiment further detected that leucine upregulated expression of DDX59 and its downstream signaling via PI3K activation. ChIP-qPCR analysis further proved the binding of DDX59 to the promoter regions of mTOR and SREBP-1c. In summary, these data prove that DDX59 positively regulates the mTOR and SREBP-1c signaling pathways leading to synthesis of milk, and leucine regulates these two signaling pathways through the PI3K-DDX59 signaling.


Assuntos
Bovinos/metabolismo , Células Epiteliais/metabolismo , Leucina/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Helicases/metabolismo , Animais , Bovinos/genética , Feminino , Fosfatidilinositol 3-Quinases/genética , RNA Helicases/genética , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
3.
Plast Reconstr Surg ; 144(1): 12-20, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31246791

RESUMO

BACKGROUND: Pathogenic mutations have been identified in approximately 10 percent of patients who present with breast cancer. Notably, failure to identify deleterious genetic mutations has particular implications for patients undergoing abdominally based breast reconstruction, as the donor site can be used only once. The authors sought to determine: (1) how many patients underwent genetic testing before unilateral abdominally based free flap breast reconstruction; (2) how often deleterious mutations were detected after abdominally based free flap breast reconstruction; and (3) the cost-effectiveness of expanding genetic testing in this patient population. METHODS: The authors retrospectively identified all patients who underwent unilateral abdominally based free flap breast reconstruction at Brigham and Women's Hospital/Dana-Farber Cancer Institute between 2007 and 2016. Chart review was performed to collect relevant demographic and clinical data. Relevant hospital financial data were obtained. RESULTS: Of the 713 who underwent free flap breast reconstruction, 160 patients met inclusion criteria, and mean follow-up was 5.8 years. Three patients (1.9 percent of 160) underwent contralateral surgery after completing reconstruction, two of whom had BRCA2 and one with ATM mutation. One hundred eleven patients met National Comprehensive Cancer Network guidelines for genetic testing, but of those only 55.9 percent (62 patients) were tested. Financial data revealed that testing every patient in the cohort would result in a net savings of $262,000. CONCLUSIONS: During a relatively short follow-up period, a small percentage of patients were diagnosed with pathogenic mutations and underwent contralateral mastectomy and reconstruction. However, because of the costliness of surgery and the decreased cost of genetic testing, it is cost-effective to test every patient before unilateral abdominally based free flap breast reconstruction.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Mutação/genética , Adulto , Idoso , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA2/genética , Neoplasias da Mama/cirurgia , Quinase do Ponto de Checagem 2/genética , Assistência à Saúde , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Retalhos de Tecido Biológico/estatística & dados numéricos , Testes Genéticos , Humanos , Mamoplastia/métodos , Mastectomia/métodos , Pessoa de Meia-Idade , RNA Helicases/genética , Estudos Retrospectivos , Ubiquitina-Proteína Ligases/genética
4.
BMC Med Genet ; 20(1): 75, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064327

RESUMO

BACKGROUND: Hereditary colon cancer is characterized by the inheritance of an abnormal gene mutation which predisposes to malignancy. Recent advances in genomic medicine have identified mutations in "novel" genes as conferring an increased risk of colorectal cancer. Mutations in the BRIP1 gene (BRCA1 Interacting Protein C- terminal helicase 1) are known to increase the risk of ovarian and breast cancers, but this genes association with colon cancer has not been previously reported. CASE PRESENTATION: We describe two patients with colon cancer whose tumor tissue were found to harbor BRIP1 mutations on analysis by next-generation sequencing. These patients were confirmed by analysis of lymphocytes to carry the mutation in the germline as well. CONCLUSIONS: These case reports highlight a previously unreported association of BRIP1 germline mutations with colon cancer predisposition.


Assuntos
Neoplasias do Colo/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Mutação em Linhagem Germinativa , RNA Helicases/genética , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem
5.
Mol Cell ; 74(5): 982-995.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076285

RESUMO

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Mitocôndrias/genética , Fatores de Iniciação de Peptídeos/genética , RNA Helicases/genética , RNA Interferente Pequeno/genética , Animais , Drosophila melanogaster/genética , Endorribonucleases/genética , Feminino , Fertilidade/genética , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Mutação , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Proteínas de Ligação a RNA/genética
6.
BMC Biol ; 17(1): 39, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088452

RESUMO

BACKGROUND: RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells. RESULTS: MOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3'-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites. CONCLUSIONS: These data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , RNA Helicases/genética , Espermatozoides/metabolismo , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , RNA Helicases/metabolismo
7.
Nat Commun ; 10(1): 1545, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948716

RESUMO

Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion.


Assuntos
DNA Helicases/fisiologia , Escherichia coli/genética , RNA Helicases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética/fisiologia , Transcrição Genética , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/metabolismo , Modelos Moleculares , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
RNA ; 25(6): 685-701, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30910870

RESUMO

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3'-5' RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/análogos & derivados , RNA Helicases DEAD-box/química , Biogênese de Organelas , RNA Helicases/química , RNA/química , Ribonucleoproteínas Nucleolares Pequenas/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por Substrato
9.
Mol Genet Genomics ; 294(4): 887-899, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30923941

RESUMO

In insects, RNAi is considered the major antiviral immune defense pathway. DsRNAs produced during viral infection are processed by Dicer enzymes into small RNAs that function as specificity determinants to silence viral genes. By contrast, in mammals, recognition of molecules associated with viral infection, such as dsRNA, by pattern recognition receptors (PRRs) initiates a signaling cascade that culminates in the production and release of signaling proteins with antiviral function such as interferons. However, in insects, the hypothesis that components of virions can be recognized as pathogen-activated molecular patterns (PAMPs) to activate the innate immune response has not been investigated systematically. In this study, the potential of VP1, that constitutes the major capsid protein of cytoplasmic polyhedrosis virus (CPV; Reoviridae), to activate a collection of immune-related genes was examined in silkworm-derived Bm5 cells. Two different methods of VP1 administration were tested, either through endogenous expression in transformed cell lines, or through addition of purified VP1-based viral-like particles to the extracellular medium. In addition, exposure to CPV virions isolated from purified polyhedra was also performed. In general, our results do not show a robust transcriptional response of immune-related genes to VP1 or CPV virions, but two exceptions were noted. First, the expression of the antimicrobial peptide (AMP) gene Attacin was strongly induced after 24 h of exposure to VP1-based VLPs. Second, the expression levels of dcr-2, an essential gene in the RNAi pathway, were greatly increased in VP1-expressing transformed Sf21 cells but not transformed Bm5 cells, indicating the existence of species-specific effects. However, the increased expression of dcr-2 did not result in increased silencing efficiency when tested in an RNAi reporter assay. Our study indicates that the capsid protein VP1 of CPV has the potential to act as a PAMP and to induce a transcriptional response in insect cells that relate both to RNAi and protein effectors such as AMPs. The identity of the PRRs and the signaling cascade that are potentially triggered by VP1 remain to be elucidated in future experiments. While this study was performed on a small scale, it can encourage more comprehensive studies with high-throughput approaches (microarray, deep sequencing) to search more systematically whether viral capsid proteins can act as PAMPs in insects and whether their production results in the induction of immune-related genes with potential antiviral function.


Assuntos
Bombyx/virologia , Proteínas do Capsídeo/imunologia , Proteínas de Insetos/genética , Reoviridae/metabolismo , Vírion/imunologia , Animais , Bombyx/genética , Bombyx/imunologia , Linhagem Celular , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade Inata , Padrões Moleculares Associados a Patógenos/imunologia , RNA Helicases/genética , Reoviridae/imunologia , Células Sf9 , Especificidade da Espécie
10.
Emerg Microbes Infect ; 8(1): 291-302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866783

RESUMO

African swine fever virus is complex DNA virus that infects pigs with mortality rates up to 100% leading to devastating socioeconomic effected in the affected countries. There is neither a vaccine nor a treatment to control ASF. African swine fever virus genome encodes two putative SF2 RNA helicases (QP509L and Q706L). In the present study, we found that these two RNA helicases do not share a common ancestral besides sharing a sequence overlap. Although, our phylogenetic studies revealed that they are conserved among virulent and non-virulent isolates, it was possible to observe a degree of variation between isolates corresponding to different genotypes occurring in distinct geographic regions. Further experiments showed that QP509L and Q706L are actively transcribed from 4 h post infection. The immunoblot analysis revealed that both protein co-localized in the viral factories at 12 h post infection, however, QP509L was also detected in the cell nucleus. Finally, siRNA assays uncover the relevant role of these proteins during viral cycle progression, in particular, for the late transcription, genome replication, and viral progeny (a reduction of infectious particles up to 99.4% when siRNA against QP509L was used and 98.4% for siRNA against Q706L). Thus, our results suggest that both helicases are essential during viral infection, highlighting the potential use of these enzymes as target for drug and vaccine development against African swine fever.


Assuntos
Vírus da Febre Suína Africana/fisiologia , Núcleo Celular/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Vírus da Febre Suína Africana/enzimologia , Vírus da Febre Suína Africana/genética , Animais , Sequência Conservada , Regulação Viral da Expressão Gênica , Filogenia , Suínos , Transcrição Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência , Replicação Viral
11.
Oncogene ; 38(22): 4340-4351, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770900

RESUMO

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a gamma-2 herpesvirus, is the causative agent of KS, primary effusion lymphoma (PEL), and a plasma cell variant of multicentric Castleman's disease. Although KSHV latency is detected in KS-related tumors, oncogenic pathways activated by KSHV latent infection are not fully understood. Here, we found that retrotransposition of long interspersed element-1 (L1), a retrotransposon in the human genome, was enhanced in PEL cells. Among the KSHV latent genes, viral FLICE-inhibitory protein (vFLIP) enhanced L1 retrotransposition in an NF-κB-dependent manner. Intracellular cell adhesion molecule-1 (ICAM-1), an NF-κB target, regulated the vFLIP-mediated enhancement of L1 retrotransposition. Furthermore, ICAM-1 downregulated the expression of Moloney leukemia virus 10 (MOV10), an L1 restriction factor. Knockdown of ICAM-1 or overexpression of MOV10 relieved the vFLIP-mediated enhancement of L1 retrotransposition. Collectively, during KSHV latency, vFLIP upregulates ICAM-1 in an NF-κB-dependent manner, which, in turn, downregulates MOV10 expression and thereby enhances L1 retrotransposition. Because active L1 retrotransposition can lead to genomic instability, which is commonly found in KS and PEL, activation of L1 retrotransposition during KSHV latency may accelerate oncogenic processes through enhancing genomic instability. Our results suggest that L1 retrotransposition may be a novel target for impeding tumor development in KSHV-infected patients.


Assuntos
Herpesvirus Humano 8/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Sarcoma de Kaposi/genética , Células 3T3 , Animais , Hiperplasia do Linfonodo Gigante/genética , Hiperplasia do Linfonodo Gigante/virologia , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação Viral da Expressão Gênica/genética , Genoma Humano/genética , Instabilidade Genômica/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , Camundongos , NF-kappa B/genética , RNA Helicases/genética , Sarcoma de Kaposi/virologia , Regulação para Cima/genética , Proteínas Virais/genética
12.
J Biol Chem ; 294(16): 6430-6438, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30804210

RESUMO

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.


Assuntos
Proteína DEAD-box 58 , DNA Helicases , Interferon beta , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose , Biossíntese de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA de Cadeia Dupla , RNA Viral , Infecções por Rhabdoviridae , Vesiculovirus , Animais , Células COS , Cercopithecus aethiops , Proteína DEAD-box 58/química , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Células RAW 264.7 , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/metabolismo , Transdução de Sinais/genética , Vesiculovirus/química , Vesiculovirus/genética , Vesiculovirus/metabolismo
13.
Gene ; 695: 18-25, 2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-30738967

RESUMO

Dengue is a severe emerging arthropod borne viral disease occurring globally. Around two fifths of the world's population, or up to 3.9 billion people, are at a risk of dengue infection. Infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection caused by other serotypes. Thus, there is a need for a vaccine which is capable of providing a life- long protection against all the serotypes of dengue virus. In our study, comparative genomics of Dengue virus (DENV) was conducted to explore potential candidates for novel vaccine targets. From our analysis we successfully found 100% conserved epitopes in Envelope protein (RCPTQGE); NS3 (SAAQRRGR, PGTSGSPI); NS4A (QRTPQDNQL); NS4B (LQAKATREAQKRA) and NS5 proteins (QRGSGQV) in all DENV serotypes. Some serotype specific conserved motifs were also found in NS1, NS5, Capsid, PrM and Envelope proteins. Using comparative genomics and immunoinformatics approach, we could find conserved epitopes which can be explored as peptide vaccine candidates to combat dengue worldwide. Serotype specific epitopes can also be exploited for rapid diagnostics. All ten proteins are explored to find the conserved epitopes in DENV serotypes, thus making it the most extensively studied viral genome so far.


Assuntos
Vírus da Dengue/imunologia , Dengue/prevenção & controle , Epitopos/imunologia , Vacinas/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Dengue/imunologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Epitopos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , RNA Helicases/genética , RNA Helicases/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Sorogrupo , Vacinas/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
14.
Nat Commun ; 10(1): 693, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30741937

RESUMO

ADP-ribosylation is a unique posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) using NAD+ as ADP-ribose donor. PARPs play an indispensable role in DNA damage repair and small molecule PARP inhibitors have emerged as potent anticancer drugs. However, to date, PARP inhibitor treatment has been restricted to patients with BRCA1/2 mutation-associated breast and ovarian cancer. One of the major challenges to extend the therapeutic potential of PARP inhibitors to other cancer types is the absence of predictive biomarkers. Here, we show that ovarian cancer cells with higher level of NADP+, an NAD+ derivative, are more sensitive to PARP inhibitors. We demonstrate that NADP+ acts as a negative regulator and suppresses ADP-ribosylation both in vitro and in vivo. NADP+ impairs ADP-ribosylation-dependent DNA damage repair and sensitizes tumor cell to chemically synthesized PARP inhibitors. Taken together, our study identifies NADP+ as an endogenous PARP inhibitor that may have implications in cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , NADP/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , ADP-Ribosilação , Animais , Biomarcadores , Linhagem Celular Tumoral/efeitos dos fármacos , Reparo do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Humanos , Camundongos , NAD/farmacologia , Neoplasias Ovarianas , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Poli ADP Ribosilação/efeitos dos fármacos , RNA Helicases/genética
15.
Proc Natl Acad Sci U S A ; 116(4): 1394-1403, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30622183

RESUMO

The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification of Vibrio cholerae tRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiI strain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence of thiI Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.


Assuntos
Endorribonucleases/genética , Complexos Multienzimáticos/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA de Transferência/genética , Archaea/genética , Bactérias/genética , Cinética , Fator Tu de Elongação de Peptídeos/genética , Controle de Qualidade , Processamento Pós-Transcricional do RNA/genética , Tiouridina/metabolismo , Vibrio cholerae/genética
16.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 360-370, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30595162

RESUMO

Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BPs, also known as Rasputin) are a family of RNA binding proteins that regulate gene expression in response to environmental stresses by controlling mRNA stability and translation. G3BPs appear to facilitate this activity through their role in stress granules for which they are considered a core component, however, it should be noted that not all stress granules contain G3BPs and this appears to be contextual depending on the environmental stress and the cell type. Although the role of G3BPs in stress granules appears to be one of its major roles, data also strongly suggests that they interact with mRNAs outside of stress granules to regulate gene expression. G3BPs have been implicated in several diseases including cancer progression, invasion, and metastasis as well as virus survival. There is now a body of evidence that suggests targeting of G3BPs could be explored as a form of cancer therapeutic. This review discusses the important discoveries and advancements made in the field of G3BPs biology over the last two decades including their roles in RNA stability, translational control of cellular transcripts, stress granule formation, cancer progression and its interactions with viruses during infection. An emerging theme for G3BPs is their ability to regulate gene expression in response to environmental stimuli, disease progression and virus infection making it an intriguing target for disease therapies.


Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , RNA Helicases/metabolismo , RNA Helicases/fisiologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/fisiologia , Estresse Fisiológico/fisiologia , Animais , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/genética , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais
17.
J Reprod Dev ; 65(2): 121-128, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30613052

RESUMO

About 10% of male infertile patients show abnormalities in spermatogenesis. The microdeletion of azoospermia factor a (AZFa) region of the Y chromosome is thought to be a cause of spermatogenic failure. However, candidate gene responsible for the spermatogenic failure in AZFa deleted patients has not been elucidated yet. Using mice, we explored the function of Ddx3y, a strong candidate gene in the Azfa region, and Ddx3x, a Ddx3y paralog on the X chromosome, in spermatogenesis. We first generated Ddx3y KO male mice using CRISPR/Cas9 and found that the Ddx3y KO male mice show normal spermatogenesis, produce morphologically normal spermatozoa, and sire healthy offspring. Because Ddx3x KO males were embryonic lethal, we next generated chimeric mice, which contain Ddx3x and Ddx3y double KO (dKO) germ cells, and found that the dKO germ cells can differentiate into spermatozoa and transmit their mutant alleles to offspring by normal mating. We conclude that Ddx3x and Ddx3y are dispensable for spermatogenesis at least in mice. Unlike human, mice have an additional Ddx3y paralog D1pas1, that has been reported to be essential for spermatogenesis. These findings suggest that human and mouse DDX3 related proteins have distinct differences in their functions.


Assuntos
Azoospermia/genética , RNA Helicases DEAD-box/genética , Fertilidade/genética , Células Germinativas/metabolismo , Antígenos de Histocompatibilidade Menor/genética , RNA Helicases/genética , Animais , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fertilização In Vitro , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/metabolismo , RNA Helicases/metabolismo , Homologia de Sequência , Espermatogênese/genética , Espermatozoides/metabolismo
18.
Biochem J ; 476(3): 467-481, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30617221

RESUMO

MOV10 has emerged as an important host antiviral factor. MOV10 not only inhibits various viruses, including human immunodeficiency virus type 1, hepatitis C virus and vesicular stomatitis virus, but also restricts the activity of retroelements long interspersed nucleotide element-1, Alu, SVA and intracisternal A particles. Here, we report that MOV10 suppresses influenza A virus infection through interacting with viral nucleoprotein (NP), sequestering viral RNP in the cytoplasm and causing the degradation of viral vRNA. The antiviral activity of MOV10 depends on the integrity of P-bodies. We also found that the antiviral activity of MOV10 is partially countered by viral NS1 protein that interferes with the interaction of MOV10 with viral NP and causes MOV10 degradation through the lysosomal pathway. Moreover, NS1-defective influenza A virus is more susceptible to MOV10 restriction. Our data not only expand the antiviral spectrum of MOV10 but also reveal the NS1 protein as the first viral antagonist of MOV10.


Assuntos
Citoplasma/metabolismo , Vírus da Influenza A/metabolismo , Proteólise , RNA Helicases/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Citoplasma/genética , Células HEK293 , Humanos , Vírus da Influenza A/genética , Lisossomos/genética , Lisossomos/metabolismo , RNA Helicases/genética , Ribonucleoproteínas/genética , Proteínas não Estruturais Virais/genética
19.
RNA ; 25(4): 407-422, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30655309

RESUMO

Nonsense-mediated mRNA decay (NMD), which is arguably the best-characterized translation-dependent regulatory pathway in mammals, selectively degrades mRNAs as a means of post-transcriptional gene control. Control can be for the purpose of ensuring the quality of gene expression. Alternatively, control can facilitate the adaptation of cells to changes in their environment. The key to NMD, no matter what its purpose, is the ATP-dependent RNA helicase upstream frameshift 1 (UPF1), without which NMD fails to occur. However, UPF1 does much more than regulate NMD. As examples, UPF1 is engaged in functionally diverse mRNA decay pathways mediated by a variety of RNA-binding proteins that include staufen, stem-loop-binding protein, glucocorticoid receptor, and regnase 1. Moreover, UPF1 promotes tudor-staphylococcal/micrococcal-like nuclease-mediated microRNA decay. In this review, we first focus on how the NMD machinery recognizes an NMD target and triggers mRNA degradation. Next, we compare and contrast the mechanisms by which UPF1 functions in the decay of other mRNAs and also in microRNA decay. UPF1, as a protein polymath, engenders cells with the ability to shape their transcriptome in response to diverse biological and physiological needs.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/genética , RNA Mensageiro/genética , Transativadores/genética , Transcriptoma , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Transativadores/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
20.
Mol Cell ; 73(4): 655-669.e7, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639244

RESUMO

In Saccharomyces cerevisiae, transcription termination at protein-coding genes is coupled to the cleavage of the nascent transcript, whereas most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3, and Sen1 (NNS). Termination involves RNA polymerase II CTD phosphorylation, but a systematic analysis of the contribution of individual residues would improve our understanding of the role of the CTD in this process. Here we investigated the effect of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this genes class. Instead, mutating Tyr1 led to widespread termination defects at non-coding genes terminating via NNS. Finally, we showed that Tyr1 is important for pausing in the 5' end of genes and that slowing down transcription suppresses termination defects. Our work highlights the importance of Tyr1-mediated pausing in NNS-dependent termination.


Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Terminação da Transcrição Genética , Sítios de Ligação , DNA Helicases/genética , Regulação Fúngica da Expressão Gênica , Mutação , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , RNA Helicases/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Tempo , Tirosina
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