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1.
Nature ; 580(7803): 391-395, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296175

RESUMO

The biological function of Z-DNA and Z-RNA, nucleic acid structures with a left-handed double helix, is poorly understood1-3. Z-DNA-binding protein 1 (ZBP1; also known as DAI or DLM-1) is a nucleic acid sensor that contains two Zα domains that bind Z-DNA4,5 and Z-RNA6-8. ZBP1 mediates host defence against some viruses6,7,9-14 by sensing viral nucleic acids6,7,10. RIPK1 deficiency, or mutation of its RIP homotypic interaction motif (RHIM), triggers ZBP1-dependent necroptosis and inflammation in mice15,16. However, the mechanisms that induce ZBP1 activation in the absence of viral infection remain unknown. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM (Ripk1mR/mR), skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1E-KO) and colitis in mice with intestinal epithelial-specific FADD deficiency (FADDIEC-KO). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that were treated with caspase inhibitors or express RIPK1 with mutated RHIM. Inhibition of nuclear export triggered the Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, which suggests that ZBP1 may recognize nuclear Z-form nucleic acids. We found that ZBP1 constitutively bound cellular double-stranded RNA in a Zα-dependent manner. Complementary reads derived from endogenous retroelements were detected in epidermal RNA, which suggests that double-stranded RNA derived from these retroelements may act as a Zα-domain ligand that triggers the activation of ZBP1. Collectively, our results provide evidence that the sensing of endogenous Z-form nucleic acids by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions-particularly in individuals with mutations in RIPK1 and CASP817-20.


Assuntos
Inflamação/metabolismo , Necroptose , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Caspase 8/metabolismo , Feminino , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Nucleicos/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia
2.
J Photochem Photobiol B ; 204: 111804, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32007677

RESUMO

The ubiquitous influence of double stranded RNAs in biological events makes them imperative to gather data based on specific binding procedure of small molecules to various RNA conformations. Particular interest may be attributed to situations wherein small molecules target RNAs altering their structures and causing functional modifications. The main focus of this study is to delve into the interactive pattern of two small molecule phenothiazinium dyes, methylene blue and new methylene blue, with three duplex RNA polynucleotides-poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C) by spectroscopic and molecular modeling techniques. Analysis of data as per Scatchard and Benesi-Hildebrand methodologies revealed highest affinity of these dyes to poly(A).poly(U) and least to poly(I).poly(C). In addition to fluorescence quenching, viscometric studies also substantiated that the dyes follow different modes of binding to different RNA polynucleotides. Distortion in the RNA structures with induced optical activity in the otherwise optically inactive dye molecules was evidenced from circular dichroism results. Dye-induced RNA structural modification occurred from extended conformation to compact particles visualized by atomic force microscopy. Molecular docking results revealed different binding patterns of the dye molecules within the RNA duplexes. The novelty of the present work lies towards a new contribution of the phenothiazinium dyes in dysfunctioning double stranded RNAs, advancing our knowledge to their potential use as RNA targeted small molecules.


Assuntos
Azul de Metileno/análogos & derivados , Azul de Metileno/química , RNA de Cadeia Dupla/química , Sítios de Ligação , Corantes/química , Azul de Metileno/metabolismo , Microscopia de Força Atômica , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Fenotiazinas/química , Poli C/química , Poli C/metabolismo , Poli G/química , Poli G/metabolismo , RNA de Cadeia Dupla/metabolismo , Espectrometria de Fluorescência , Espectrofotometria , Viscosidade
3.
Nat Commun ; 11(1): 799, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034135

RESUMO

RNA editing and splicing are the two major processes that dynamically regulate human transcriptome diversity. Despite growing evidence of crosstalk between RNA editing enzymes (mainly ADAR1) and splicing machineries, detailed mechanistic explanations and their biological importance in diseases, such as cancer are still lacking. Herein, we identify approximately a hundred high-confidence splicing events altered by ADAR1 and/or ADAR2, and ADAR1 or ADAR2 protein can regulate cassette exons in both directions. We unravel a binding tendency of ADARs to dsRNAs that involves GA-rich sequences for editing and splicing regulation. ADAR1 edits an intronic splicing silencer, leading to recruitment of SRSF7 and repression of exon inclusion. We also present a mechanism through which ADAR2 binds to dsRNA formed between GA-rich sequences and polypyrimidine (Py)-tract and precludes access of U2AF65 to 3' splice site. Furthermore, we find these ADARs-regulated splicing changes per se influence tumorigenesis, not merely byproducts of ADARs editing and binding.


Assuntos
Adenosina Desaminase/metabolismo , Neoplasias/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Processamento Alternativo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos Endogâmicos NOD , Degradação do RNAm Mediada por Códon sem Sentido , Edição de RNA , Sítios de Splice de RNA , Processamento de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética
4.
Appl Microbiol Biotechnol ; 104(5): 2125-2135, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932895

RESUMO

Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3' ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. • A more consistent effect was observed for methylated siRNAs. • The findings are important for development of plant gene regulation approaches.


Assuntos
Regulação da Expressão Gênica de Plantas , Inativação Gênica , RNA Interferente Pequeno/genética , Transgenes/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Metilação de DNA , Regulação da Expressão Gênica de Plantas/genética , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Genética
5.
Exp Parasitol ; 209: 107829, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31887531

RESUMO

Ticks, obligatory blood-feeding arthropods, are a major pathogen vector in humans and animals worldwide. Anti-tick vaccines are an exciting alternative to chemical acaricides for controlling these disease-transmitting vectors. However, identification of protective antigens for anti-tick vaccine development is challenging. Different ribosomal proteins play multifunctional roles in tick survival and feeding. Here, we first report the cloning and molecular characterization of ribosomal protein S27 (RPS-27) from the hard tick Haemaphysalis longicornis. We identified a complete open reading frame (ORF) of RPS-27: a 255-bp (base pair) cDNA encoding a mature protein of 84 amino-acid residues with a 9.4-kDa predicted molecular mass. Amino-acid sequence analysis revealed that RPS-27 was highly conserved among different tick and vertebrate animals with identity ranges of 97-98% and 60-85%, respectively. Phylogenetic tree analysis showed that RPS-27 from different tick species clustered together. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the RPS-27 mRNA transcript was expressed in all life stages. At the tissue level, it was more highly expressed in the salivary gland than in the midgut for both the fed and unfed conditions, which indicates a role for RPS-27 in tick feeding. In vitro analysis showed that recombinant RPS-27 (10-RPS-27) was successfully expressed in a pGEMEX-2 vector with an estimated 45-kDa molecular mass. The functional importance of RPS-27 was determined by gene silencing through RNA interference (RNAi). RPS-27 silencing showed a significant (P < 0.05) reduction of feeding abilityand engorgement weight after the blood meal in both nymph and adult female ticks and also significantly (P < 0.05) reduced molting rate in nymph. In addition, RPS-27 silencing in eggs led to abnormalities in shape and hatching. Taken together, our results suggest that RPS-27 is an important molecule that plays multiple roles in the tick life cycle including in both feeding and reproduction. Therefore, RPS-27 is an exciting target for future tick control strategies.


Assuntos
Inativação Gênica , Ixodidae/genética , Fases de Leitura Aberta , Proteínas Ribossômicas/genética , Vacinas/genética , Sequência de Aminoácidos , Análise de Variância , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Expressão Gênica , Ixodidae/classificação , Ixodidae/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Ribossômicas/química , Proteínas Ribossômicas/imunologia , Alinhamento de Sequência , Doenças Transmitidas por Carrapatos/prevenção & controle , Doenças Transmitidas por Carrapatos/transmissão , Transcrição Genética
6.
J Chem Ecol ; 46(2): 138-149, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31853816

RESUMO

Chemosensory proteins (CSPs) are thought to play roles in the insect olfactory system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. The band-winged grasshopper, Oedaleus asiaticus Bei-Bienko, is one of the most important grasshopper pests in northern China, but there is little information about its olfactory system. In order to investigate the olfactory functions of CSPs in this pest, three CSP genes (OasiCSP4, OasiCSP11 and OasiCSP12) were expressed in Escherichia coli, and the binding affinities of the three recombinant CSP proteins were measured for 16 volatiles from the host plant (Stipa krylovii), fecal material and body of live adult O. asiaticus using fluorescence competitive binding assays. To further verify their olfactory functions, RNA interference (RNAi) and electrophysiological recording were conducted. The three recombinant proteins displayed different degrees of binding to various volatiles in ligand-binding assays, with OasiCSP12 having higher binding affinities for more volatiles than OasiCSP4 and OasiCSP11. OasiCSP12 exhibited strong binding affinities (Ki < 20 µΜ) for five host plant volatiles and one volatile from the live body of adult O. asiaticus. The transcript levels of the three OasiCSP genes were significantly lower after silencing the individual genes by RNAi, which in turn reduced the EAG responses in adults of both sexes to most tested compounds. Our study indicates that these three OasiCSPs are involved in the detection of volatile semiochemicals, and may play important roles in finding host plants and in aggregation in O. asiaticus.


Assuntos
Gafanhotos/metabolismo , Proteínas de Insetos/metabolismo , Receptores Odorantes/metabolismo , Animais , Ligação Competitiva , Feminino , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Masculino , Poaceae/química , Poaceae/metabolismo , Ligação Proteica , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Receptores Odorantes/antagonistas & inibidores , Receptores Odorantes/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo
7.
Adv Exp Med Biol ; 1172: 157-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628656

RESUMO

RIG-I-like receptors (RLRs) are an important family of pattern recognition receptors. They activate the immunological responses against viral infections by sensing RNAs in the cytoplasm. Recent studies provide significant insights into the activation and transduction mechanisms of RLRs family (members including RIG-I, MDA5, and LGP2). Here we review our current understanding of the structures of RLRs. Structural characterizations of RLRs family have revealed the mechanism of their actions at molecular level. The activation mechanisms of RIG-I and MDA5 are different, despite both of them have similar domain organizations and bind to dsRNA ligands. RIG-I, but not MDA5, adopts an auto-suppression conformation in the absence of RNA. In addition to ligand triggered receptor oligomerization, the activities of these receptors are also regulated by several posttranslational modifications, especially ubiquitination. Overall, these structural studies play critical roles in promoting the understanding of viral RNA recognition mechanisms by the host innate immune system, which also contribute to the designing of drugs for treatment of viral infection. However, much work remains to be done in studying the signaling pathway of MDA5 and LGP2, particularly by structural biology.


Assuntos
Proteína DEAD-box 58 , RNA Helicases DEAD-box , Imunidade Inata , RNA Viral , Animais , Proteína DEAD-box 58/química , Proteína DEAD-box 58/metabolismo , Humanos , Helicase IFIH1 Induzida por Interferon , RNA de Cadeia Dupla/metabolismo , RNA Viral/análise , RNA Viral/metabolismo , Transdução de Sinais , Viroses/imunologia
8.
PLoS Pathog ; 15(10): e1008111, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626661

RESUMO

The herpes simplex virus virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs and blunts host innate antiviral responses. Previous work demonstrated that cells infected with vhs mutants display enhanced activation of the host double-stranded RNA (dsRNA)-activated protein kinase R (PKR), implying that vhs limits dsRNA accumulation in infected cells. Confirming this hypothesis, we show that partially complementary transcripts of the UL23/UL24 and UL30/31 regions of the viral genome increase in abundance when vhs is inactivated, giving rise to greatly increased levels of intracellular dsRNA formed by annealing of the overlapping portions of these RNAs. Thus, vhs limits accumulation of dsRNA at least in part by reducing the levels of complementary viral transcripts. We then asked if vhs also destabilizes dsRNA after its initial formation. Here, we used a reporter system employing two mCherry expression plasmids bearing complementary 3' UTRs to produce defined dsRNA species in uninfected cells. The dsRNAs are unstable, but are markedly stabilized by co-expressing the HSV dsRNA-binding protein US11. Strikingly, vhs delivered by super-infecting HSV virions accelerates the decay of these pre-formed dsRNAs in both the presence and absence of US11, a novel and unanticipated activity of vhs. Vhs binds the host RNA helicase eIF4A, and we find that vhs-induced dsRNA decay is attenuated by the eIF4A inhibitor hippuristanol, providing evidence that eIF4A participates in the process. Our results show that a herpesvirus host shutoff RNase destabilizes dsRNA in addition to targeting partially complementary viral mRNAs, raising the possibility that the mRNA destabilizing proteins of other viral pathogens dampen the host response to dsRNA through similar mechanisms.


Assuntos
Estabilidade de RNA/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribonucleases/metabolismo , Simplexvirus/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , DNA Polimerase Dirigida por DNA/metabolismo , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4F em Eucariotos/metabolismo , Exodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Vero
9.
Mol Cell ; 75(6): 1218-1228.e6, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31494033

RESUMO

Viral and endogenous double-stranded RNA (dsRNA) is a potent trigger for programmed RNA degradation by the 2-5A/RNase L complex in cells of all mammals. This 2-5A-mediated decay (2-5AMD) is a conserved stress response switching global protein synthesis from homeostasis to production of interferons (IFNs). To understand this mechanism, we examined 2-5AMD in human cells and found that it triggers polysome collapse characteristic of inhibited translation initiation. We determined that translation initiation complexes and ribosomes purified from translation-arrested cells remain functional. However, spike-in RNA sequencing (RNA-seq) revealed cell-wide decay of basal mRNAs accompanied by rapid accumulation of mRNAs encoding innate immune proteins. Our data attribute this 2-5AMD evasion to better stability of defense mRNAs and positive feedback in the IFN response amplified by RNase L-resistant molecules. We conclude that 2-5AMD and transcription act in concert to refill mammalian cells with defense mRNAs, thereby "prioritizing" the synthesis of innate immune proteins.


Assuntos
Endorribonucleases/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Transcrição Genética , Células A549 , Endorribonucleases/genética , Humanos , Imunidade Inata , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética
10.
Mol Cell ; 75(6): 1203-1217.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31494035

RESUMO

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-ß. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.


Assuntos
Reprogramação Celular , Endorribonucleases/metabolismo , Interferon beta/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , eIF-2 Quinase/metabolismo , Células A549 , Endorribonucleases/genética , Células HEK293 , Humanos , Interferon beta/genética , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , eIF-2 Quinase/genética
11.
Appl Microbiol Biotechnol ; 103(20): 8485-8496, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31486873

RESUMO

Double-stranded RNA (dsRNA) inducing RNA interference (RNAi) is expected to be applicable to management of agricultural pests. In this study, we selected a ladybird beetle, Henosepilachna vigintioctopunctata, as a model target pest that devours vegetable leaves, and examined the effects of feeding the pest sterilized microbes highly accumulating a target dsRNA for RNAi induction. We constructed an efficient production system for diap1*-dsRNA, which suppresses expression of the essential gene diap1 (encoding death-associated inhibitor of apoptosis protein 1) in H. vigintioctopunctata, using an industrial strain of Corynebacterium glutamicum as the host microbe. The diap1*-dsRNA was overproduced in C. glutamicum by convergent transcription using a strong promoter derived from corynephage BFK20, and the amount of dsRNA accumulated in fermented cells reached about 75 mg per liter of culture. In addition, we developed a convenient method for stabilizing the dsRNA within the microbes by simply sterilizing the diap1*-dsRNA-expressing C. glutamicum cells with ethanol. When the sterilized microbes containing diap1*-dsRNA were fed to larvae of H. vigintioctopunctata, diap1 expression in the pest was suppressed, and the leaf-feeding activity of the larvae declined. These results suggest that this system is capable of producing stabilized dsRNA for RNAi at low cost, and it will open a way to practical application of dsRNA as an environmentally-friendly agricultural insecticide.


Assuntos
Besouros/microbiologia , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/genética , Inseticidas/metabolismo , Controle Biológico de Vetores/métodos , RNA de Cadeia Dupla/metabolismo , Animais , Besouros/crescimento & desenvolvimento , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Larva/crescimento & desenvolvimento , Larva/microbiologia , Interferência de RNA
12.
J Chem Ecol ; 45(10): 849-857, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31512099

RESUMO

Odorant binding proteins (OBPs) play a key role in chemoreception in insects. In an earlier study, we identified CmedOBP14 from the rice leaf folder, Cnaphalocrocis medinalis, with potential physiological functions in olfaction. Here, we performed a competitive binding assay under different pH conditions as well as knockdown via RNA interference to determine the specific role of CmedOBP14 in C. medinalis. CmedOBP14 displayed broad binding affinities to many host-related compounds, with higher affinities at pH 7.4 compared with pH 5.0. After treatment with CmedOBP14-dsRNA, the transcript level of OBP14 was significantly decreased at 72 h compared with controls, and the electroantennogram response evoked by nerolidol, L-limonene and beta-ionone was reduced. Furthermore, behavioral assays revealed consistent patterns among these compounds, especially for nerolidol, with adults could no longer able to differentiate 0.1% nerolidol from controls. RNAi experiments suggest that at least in part, CmedOBP14 mediates the ability to smell nerolidol and beta-ionone.


Assuntos
Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Receptores Odorantes/metabolismo , Animais , Antenas de Artrópodes/metabolismo , Comportamento Animal/efeitos dos fármacos , Ligação Competitiva , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Norisoprenoides/farmacologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Receptores Odorantes/antagonistas & inibidores , Receptores Odorantes/genética , Sesquiterpenos/farmacologia
13.
Biochemistry (Mosc) ; 84(8): 896-904, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522671

RESUMO

RNA editing by adenosine deaminases of the ADAR family attracts a growing interest of researchers, both zoologists studying ecological and evolutionary plasticity of invertebrates and medical biochemists focusing on the mechanisms of cancer and other human diseases. These enzymes deaminate adenosine residues in the double-stranded (ds) regions of RNA with the formation of inosine. As a result, some RNAs change their three-dimensional structure and functions. Adenosine-to-inosine editing in the mRNA coding sequences may cause amino acid substitutions in the encoded proteins. Here, we reviewed current concepts on the functions of two active ADAR isoforms identified in mammals (including humans). The ADAR1 protein, which acts non-specifically on extended dsRNA regions, is capable of immunosuppression via inactivation of the dsRNA interactions with specific sensors inducing the cell immunity. Expression of a specific ADAR1 splicing variant is regulated by the type I interferons by the negative feedback mechanism. It was shown that immunosuppressing effects of ADAR1 facilitate progression of some types of cancer. On the other hand, changes in the amino acid sequences resulting from the mRNA editing by the ADAR enzymes can result in the formation of neoantigens that can activate the antitumor immunity. The ADAR2 isoform acts on RNA more selectively; its function is associated with the editing of mRNA coding regions and can lead to the amino acid substitutions, in particular, those essential for the proper functioning of some neurotransmitter receptors in the central nervous system.


Assuntos
Adenosina Desaminase/metabolismo , Carcinogênese/metabolismo , Plasticidade Neuronal/fisiologia , Edição de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/imunologia , Sequência de Aminoácidos , Animais , Senescência Celular/fisiologia , Humanos , Inosina/metabolismo , Interferon Tipo I/metabolismo , Proteoma/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/imunologia
14.
Oxid Med Cell Longev ; 2019: 2860642, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379987

RESUMO

Phosphatidylcholine is one of the major phospholipids comprising cellular membrane and is known to have several health-promoting activities, including the improvement of brain function and liver repair. In this paper, we examine the in vivo effect of dietary supplementation with phosphatidylcholine on the response to environmental stressors and aging in C. elegans. Treatment with phosphatidylcholine significantly increased the survival of worms under oxidative stress conditions. However, there was no significant difference in response to stresses caused by heat shock or ultraviolet irradiation. Oxidative stress is believed to be one of the major causal factors of aging. Then, we examined the effect of phosphatidylcholine on lifespan and age-related physiological changes. Phosphatidylcholine showed a lifespan-extending effect and a reduction in fertility, possibly as a tradeoff for long lifespan. Age-related decline of motility was also significantly delayed by supplementation with phosphatidylcholine. Interestingly, the expressions of well-known longevity-assuring genes, hsp-16.2 and sod-3, were significantly upregulated by dietary intervention with phosphatidylcholine. DAF-16, a transcription factor modulating stress response genes, was accumulated in the nucleus by phosphatidylcholine treatment. Increase of the ROS level with phosphatidylcholine suggests that the antioxidant and lifespan-extending effects are due to the hormetic effect of phosphatidylcholine. Phosphatidylcholine also showed a protective effect against amyloid beta-induced toxicity in Alzheimer's disease model animals. Experiments with long-lived mutants revealed that the lifespan-extending effect of phosphatidylcholine specifically overlapped with that of reduced insulin/IGF-1-like signaling and required DAF-16. These findings showed the antioxidant and antiaging activities of phosphatidylcholine for the first time in vivo. Further studies focusing on the identification of underlying cellular mechanisms involved in the antiaging effect will increase the possibility of using phosphatidylcholine for the development of antiaging therapeutics.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Longevidade/efeitos dos fármacos , Fosfatidilcolinas/farmacologia , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos
15.
Nucleic Acids Res ; 47(17): 9104-9114, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31372641

RESUMO

Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3' regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Inativação Gênica , RNA Replicase/genética , Proteínas de Arabidopsis/metabolismo , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Genéticos , Plantas Geneticamente Modificadas/genética , RNA Replicase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo
16.
PLoS Genet ; 15(7): e1008240, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31365523

RESUMO

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Poliadenilação , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Estabilidade de RNA , RNA Antissenso/química , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo
17.
Nucleic Acids Res ; 47(16): 8693-8707, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31361901

RESUMO

Zika virus is a positive single-strand RNA virus whose replication involved RNA unwinding and synthesis. ZIKV NS3 contains a helicase domain, but its enzymatic activity is not fully characterized. Here, we established a dsRNA unwinding assay based on the FRET effect to study the helicase activity of ZIKV NS3, which provided kinetic information in real time. We found that ZIKV NS3 specifically unwound dsRNA/dsDNA with a 3' overhang in the 3' to 5' direction. The RNA unwinding ability of NS3 significantly decreased when the duplex was longer than 18 base pairs. The helicase activity of NS3 depends on ATP hydrolysis and binding to RNA. Mutations in the ATP binding region or the RNA binding region of NS3 impair its helicase activity, thus blocking viral replication in the cell. Furthermore, we showed that ZIKV NS5 interacted with NS3 and stimulated its helicase activity. Disrupting NS3-NS5 interaction resulted in a defect in viral replication, revealing the tight coupling of RNA unwinding and synthesis. We suggest that NS3 helicase activity is stimulated by NS5; thus, viral replication can be carried out efficiently. Our work provides a molecular mechanism of ZIKV NS3 unwinding and novel insights into ZIKV replication.


Assuntos
Regulação Viral da Expressão Gênica , RNA de Cadeia Dupla/química , RNA Viral/química , Proteínas não Estruturais Virais/química , Zika virus/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Cricetulus , Células Epiteliais/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Zika virus/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-31298608

RESUMO

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N6-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N6-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.


Assuntos
Adenosina/análogos & derivados , Interferência de RNA/imunologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Adenosina/química , Sequência de Bases , Expressão Gênica , Células HeLa , Humanos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo
19.
Nat Commun ; 10(1): 3085, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300642

RESUMO

DEAD-box helicases (DDXs) regulate RNA processing and metabolism by unwinding short double-stranded (ds) RNAs. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:single-stranded RNA complex, and the structure of a D2:dsRNA complex that is thought to represent a pre-unwound state. However, the structure of a pre-unwound D1D2:dsRNA complex remains elusive, and thus, the mechanism of DDX action is not fully understood. Here, we describe the structure of a D1D2 core in complex with a 23-base pair dsRNA at pre-unwound state, revealing that two DDXs recognize a 2-turn dsRNA, each DDX mainly recognizes a single RNA strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner.


Assuntos
RNA Helicases DEAD-box/ultraestrutura , RNA de Cadeia Dupla/metabolismo , RNA Helicases DEAD-box/isolamento & purificação , RNA Helicases DEAD-box/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Difração de Raios X
20.
J Biol Chem ; 294(28): 11047-11053, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31177094

RESUMO

A key metabolic adaptation of some species that face hypoxia as part of their life cycle involves an alternative electron transport chain in which rhodoquinone (RQ) is required for fumarate reduction and ATP production. RQ biosynthesis in bacteria and protists requires ubiquinone (Q) as a precursor. In contrast, Q is not a precursor for RQ biosynthesis in animals such as parasitic helminths, and most details of this pathway have remained elusive. Here, we used Caenorhabditis elegans as a model animal to elucidate key steps in RQ biosynthesis. Using RNAi and a series of C. elegans mutants, we found that arylamine metabolites from the kynurenine pathway are essential precursors for RQ biosynthesis de novo Deletion of kynu-1, encoding a kynureninase that converts l-kynurenine (KYN) to anthranilic acid (AA) and 3-hydroxykynurenine (3HKYN) to 3-hydroxyanthranilic acid (3HAA), completely abolished RQ biosynthesis but did not affect Q levels. Deletion of kmo-1, which encodes a kynurenine 3-monooxygenase that converts KYN to 3HKYN, drastically reduced RQ but not Q levels. Knockdown of the Q biosynthetic genes coq-5 and coq-6 affected both Q and RQ levels, indicating that both biosynthetic pathways share common enzymes. Our study reveals that two pathways for RQ biosynthesis have independently evolved. Unlike in bacteria, where amination is the last step in RQ biosynthesis, in worms the pathway begins with the arylamine precursor AA or 3HAA. Because RQ is absent in mammalian hosts of helminths, inhibition of RQ biosynthesis may have potential utility for targeting parasitic infections that cause important neglected tropical diseases.


Assuntos
Caenorhabditis elegans/metabolismo , Cinurenina/metabolismo , Ubiquinona/análogos & derivados , Animais , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrolases/antagonistas & inibidores , Hidrolases/genética , Hidrolases/metabolismo , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Quinurenina 3-Mono-Oxigenase/genética , Quinurenina 3-Mono-Oxigenase/metabolismo , Espectrometria de Massas , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Tela Subcutânea/metabolismo , Ubiquinona/análise , Ubiquinona/biossíntese , Ubiquinona/metabolismo
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