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1.
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
2.
Arch Virol ; 165(1): 227-231, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31659444

RESUMO

Three viral contig sequences, which represented complete genome of a novel virus with three dsRNAs of 1,712 nucleotides (nt) (dsRNA1), 1,504 nt (dsRNA2) and 1,353 nt (dsRNA3), were found in tea-oil camellia plants by high-throughput sequencing analysis. The three dsRNAs were re-sequenced by RT-PCR cloning. The largest dsRNA, dsRNA1, had a single open reading frame (ORF) that encoded a putative 52.7-kDa protein of a putative viral RNA-dependent RNA polymerase (RdRp). DsRNA2 and dsRNA3 were predicted to encode putative capsid proteins (CPs) of 40.47 kDa and 40.59 kDa, respectively. The virus, which is provisionally named "tea-oil camellia deltapartitivirus 1",  shared amino acid sequence itentities of 36.09-69.18% with members of the genus Deltapartitivirus on RdRp. Phylogenetic analysis based on RdRp also placed the new virus and other deltapartitiviruses together in a group, suggesting that this virus should be considered a new member of the genus Deltapartitivirus.


Assuntos
Camellia/virologia , Vírus de RNA/genética , Sequenciamento Completo do Genoma/métodos , Mapeamento de Sequências Contíguas , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fases de Leitura Aberta , Filogenia , Vírus de RNA/classificação , RNA de Cadeia Dupla/genética
3.
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
4.
Nucleic Acids Res ; 48(4): 1811-1827, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31872227

RESUMO

Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.


Assuntos
Proteínas Argonauta/genética , Proteínas de Caenorhabditis elegans/genética , RNA Interferente Pequeno/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica , Células Germinativas/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , Transcriptoma/genética
5.
Nucleic Acids Res ; 48(4): 2091-2106, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31875226

RESUMO

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the ß1-ß2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.


Assuntos
Fator 1 de Ribosilação do ADP/química , Proteínas do Citoesqueleto/química , Motivo de Ligação ao RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/química , Proteínas de Ligação a RNA/química , Fator 1 de Ribosilação do ADP/genética , Sítios de Ligação/genética , Citoplasma/química , Citoplasma/genética , Proteínas do Citoesqueleto/genética , Humanos , Conformação Proteica , Estabilidade de RNA/genética , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética
6.
Nat Struct Mol Biol ; 26(11): 1023-1034, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695188

RESUMO

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8-3.5 Å) RdRp-RNA in situ structures-representing quiescent, initiation, early elongation, elongation and abortive states-under seven experimental conditions of CPV. We observed the 'Y'-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.


Assuntos
RNA de Cadeia Dupla/genética , RNA Viral/genética , Reoviridae/genética , Transcrição Genética , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , RNA Replicase/química , RNA Replicase/ultraestrutura , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/ultraestrutura , RNA Viral/química , RNA Viral/ultraestrutura , Reoviridae/química , Reoviridae/ultraestrutura , Infecções por Reoviridae/virologia , Proteínas Virais/química , Proteínas Virais/ultraestrutura
7.
Parasitol Res ; 118(12): 3565-3570, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31701295

RESUMO

The flagellated protozoon Trichomonas vaginalis, responsible for trichomoniasis, can establish a symbiotic relationship with the bacterium Mycoplasma hominis and can harbor double-stranded RNA Trichomonasvirus (TVV). In this study, we investigated by real-time PCR the prevalence of the four TVVs and of M. hominis among 48 T. vaginalis strains isolated in Italy, and we evaluated a possible association with metronidazole resistance. Fifty percent of the analyzed trichomonad strains tested positive for at least one TVV T. vaginalis, with TVV2 being the most prevalent, followed by TVV1 and TVV3. Two T. vaginalis strains were infected by TVV4, detected in Europe for the first time. Interestingly, we found more than one TVV species in 75% of positive trichomonad strains. M. hominis was present in 81.25% of T. vaginalis isolates tested, and no statistically significant association was observed with the infection by any TVV. Metronidazole sensitivity of T. vaginalis isolates was evaluated in vitro, and no correlation was observed between minimal lethal concentration and the presence of TVVs. This is the first report on TVV infection of T. vaginalis in Italy. Even if no association of TVV positive isolates with the presence of the symbiont M. hominis or with metronidazole resistance was observed, further studies are needed to shed light on the effective role of infecting microorganisms on the pathophysiology of T. vaginalis.


Assuntos
Mycoplasma hominis/isolamento & purificação , Vírus de RNA/isolamento & purificação , Trichomonas vaginalis/microbiologia , Trichomonas vaginalis/virologia , Antiprotozoários/farmacologia , Resistência a Medicamentos , Humanos , Itália , Metronidazol/farmacologia , Mycoplasma hominis/classificação , Mycoplasma hominis/genética , Mycoplasma hominis/fisiologia , Prevalência , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/fisiologia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Simbiose , Tricomoníase/parasitologia , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/fisiologia
8.
Nat Cell Biol ; 21(11): 1346-1356, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685995

RESUMO

In the past two decades, emerging studies have suggested that DExD/H box helicases belonging to helicase superfamily 2 (SF2) play essential roles in antiviral innate immunity. However, the antiviral functions of helicase SF1, which shares a conserved helicase core with SF2, are little understood. Here we demonstrate that zinc finger NFX1-type containing 1 (ZNFX1), a helicase SF1, is an interferon (IFN)-stimulated, mitochondrial-localised dsRNA sensor that specifically restricts the replication of RNA viruses. Upon virus infection, ZNFX1 immediately recognizes viral RNA through its Armadillo-type fold and P-loop domain and then interacts with mitochondrial antiviral signalling protein to initiate the type I IFN response without depending on retinoic acid-inducible gene I-like receptors (RLRs). In short, as is the case with interferon-stimulated genes (ISGs) alone, ZNFX1 can induce IFN and ISG expression at an early stage of RNA virus infection to form a positively regulated loop of the well-known RLR signalling. This provides another layer of understanding of the complexity of antiviral immunity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos de Neoplasias/genética , Mitocôndrias/imunologia , Fatores de Processamento de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Vesiculovirus/genética , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/virologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/virologia , Conformação de Ácido Nucleico , Poli I-C/farmacologia , Cultura Primária de Células , Ligação Proteica , Fatores de Processamento de RNA/imunologia , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/imunologia , RNA Viral/química , RNA Viral/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/imunologia
9.
Virus Genes ; 55(6): 854-858, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31605266

RESUMO

A virus isolate from tabasco pepper (Capsicum frutescens) has been reported as a strain of the comovirus Andean potato mottle virus (APMoV). Using the replicative intermediate viral dsRNA, the pepper virus strain was sequenced by Illumina MiSeq. The viral genome was de novo assembled resulting in two RNAs with lengths of 6028 and 3646 nt. Nucleotide sequence analysis indicated that they corresponded to the RNA-1 and RNA-2 of a novel comovirus which we tentatively named pepper mild mosaic virus (PepMMV). Predictions of the open reading frame (ORF) of RNA-1 resulted in a single ORF of 5871 nt with five cistrons typical of comoviruses, cofactor proteinase, helicase, viral protein genome-linked, 3C-like proteinase (Pro), and RNA-dependent RNA polymerase (RdRP). Similarly, sequence analysis of RNA-2 resulted in a single ORF of 3009 nt with two cistrons typical of comoviruses: movement protein and coat protein (large coat protein and small coat proteins). In pairwise amino acid sequence alignments using the Pro-Pol protein, PepMMV shared the closest identities with broad bean true mosaic virus and cowpea mosaic virus, 56% and 53.9% respectively. In contrast, in alignments of the amino acid sequence of the coat protein (small and large coat proteins) PepMMV shared the closest identities to APMoV and red clover mottle virus, 54% and 40.9% respectively. A phylogenetic tree constructed using the conserved domains for the Pro-Pol from all members of the family Secoviridae confirmed the comovirus nature of the virus. Phylogenetic and sequence analyses supports proposing PepMMV as a new species of the genus Comovirus.


Assuntos
Comovirus/genética , Genoma Viral/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos/genética , Capsicum/genética , Capsicum/virologia , Anotação de Sequência Molecular , Vírus do Mosaico/genética , Fases de Leitura Aberta/genética , Filogenia , RNA Replicase/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética
10.
Nucleic Acids Res ; 47(19): 10059-10071, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501873

RESUMO

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Inativação Gênica , RNA Replicase/genética , RNA de Cadeia Dupla/genética , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/genética , Linhagem da Célula/genética , Células Germinativas , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mosaicismo , Neurônios/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
11.
Nat Commun ; 10(1): 4033, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562302

RESUMO

Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Potyvirus/genética , Soja/imunologia , RNA Polimerases Dirigidas por DNA/imunologia , Resistência à Doença/genética , Genes de Plantas , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/imunologia , Potyvirus/imunologia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Soja/genética , Soja/virologia , Replicação Viral/imunologia
12.
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
13.
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
14.
Int J Mol Sci ; 20(17)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484427

RESUMO

Studying sex communication is necessary to develop new methods to control the population expansion of gonochoristic species Bursaphelenchus xylophilus, the pathogen of pine wilt disease (PWD). Small chemical signals called ascarosides have been reported to attract potential mates. However, they have not been studied in the sex attraction of B. xylophilus. Here, we confirmed the sex attraction of B. xylophilus using a chemotaxis assay. Then, we cloned the downstream ascaroside biosynthetic gene Bx-daf-22 and explored its function in the sex attraction of B. xylophilus through bioinformatics analysis and RNA interference. The secretions of females and males were the sources of sex attraction in B. xylophilus, and the attractiveness of females to males was stronger than that of males to females. Compared with daf-22 of Caenorhabditis elegans, Bx-daf-22 underwent gene duplication events, resulting in Bx-daf-22.1, Bx-daf-22.2, and Bx-daf-22.3. RNA interference revealed that the attractiveness of female secretions to males increased after all three Bx-daf-22 genes or Bx-daf-22.3 had been interfered. However, the reciprocal experiments had no effect on the attractiveness of male secretions to females. Thus, Bx-daf-22 genes, especially Bx-daf-22.3, may be crucial for the effectiveness of female sex attractants. Our studies provide fundamental information to help identify the specific components and signal pathways of sex attractants in B. xylophilus.


Assuntos
Tylenchida/genética , Animais , Feminino , Masculino , Interferência de RNA , RNA de Cadeia Dupla/genética
15.
Mol Cell ; 75(3): 576-589.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398324

RESUMO

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.


Assuntos
Proteínas de Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Replicase/genética , Ribonuclease III/genética , Transcrição Genética , Arabidopsis/genética , Proteínas Argonauta/genética , Metilação de DNA/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Transdução de Sinais
16.
Arch Virol ; 164(10): 2585-2592, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377889

RESUMO

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.


Assuntos
Anguilla/virologia , Doenças dos Peixes/virologia , Infecções por Reoviridae/veterinária , Reoviridae/isolamento & purificação , Animais , Encéfalo/virologia , Linhagem Celular , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia Eletrônica de Transmissão , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Reoviridae/classificação , Reoviridae/genética , Infecções por Reoviridae/virologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Coloração e Rotulagem , Proteínas Virais/genética , Vírion/ultraestrutura , Cultura de Vírus
17.
Gene ; 719: 144074, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31446094

RESUMO

Kinesin-14 motor es-kifc1 is highly expressed in the male reproductive system of the Chinese mitten crab Eriocheir sinensis (E. sinensis). In addition to acrosomal formation, es-KIFC1 also tightly surrounds the nucleus and its specific mechanism remains unknown. During spermatogenesis, sperm nucleus dents into a cup-shaped structure with several radial arms and completed the nuclear decondensation. In this study, the spatial expression pattern of es-KIFC1 indicates a potential function in nuclear formation with the nuclear localization sequence (NLS) on N-terminal domain which is crucial for the translocation of es-KIFC1 into the nucleus. The Motor domain is associated with microtubule modulation and the Golgi vesicles positioning. Furthermore, the expression level of es-KIFC1 is not only related to the seasonal variation of crustacean development, but also associates with mature sperm storage. The double strand RNA (dsRNA) mediated RNA interference manifests that the cup-shaped sperm nucleus is remarkably malformed and even separates the chromatin throughout the nuclei at the last stage of spermiogenesis. Besides, the sperm nucleus almost disperses its structure and separates the chromatin into several segments throughout the nucleus showing an asymmetrical performance without cytoskeleton. In summary, these results indicate the importance of es-KIFC1 in microtubule positioning and the maintenance of the mature sperm nuclei.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/fisiologia , Núcleo Celular/metabolismo , Cinesina/genética , Espermatogênese/genética , Animais , Proteínas de Artrópodes/metabolismo , Núcleo Celular/ultraestrutura , Citoesqueleto/metabolismo , Cinesina/metabolismo , Masculino , Microtúbulos/metabolismo , Transporte Proteico , RNA de Cadeia Dupla/genética , Espermatozoides/ultraestrutura
18.
Pestic Biochem Physiol ; 159: 85-90, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400788

RESUMO

RNA interference (RNAi) is a potentially useful pest control method because of its high specificity. Silencing the expression of important RNAi target genes of pests will block important biological processes and reduce pest damage. Ecdysone is a unique arthropod hormone and the ecdysone receptor (EcR) is a key factor in molting pathway. We investigated the possibility that dsRNA targeting of the EcR of Tetranychus cinnabarinus (TcEcR) could effectively block development from larvae to adults. The mRNA level of TcEcR was highest in the larva stage, and 73.1% of the mites failed to survive the larva stage when TcEcR expression was silenced. Only 11.7% of T. cinnabarinus ingesting dsRNA successfully developed into adults, while 86.7% in the control succeeded in molting across each stage. RNAi significantly increased the developmental intervals of T. cinnabarinus. Under the effects of dsRNA, development times for the larva and first nymph doubled. Phenotype of body size change and death were observed during the development of T. cinnabarinus ingesting dsRNA. These findings suggest that RNAi is a potential means for the control of T. cinnabarinus. Genes in hormone pathways such as EcR are possible RNAi targets.


Assuntos
Larva/metabolismo , Interferência de RNA/fisiologia , Receptores de Esteroides/metabolismo , Tetranychidae/metabolismo , Animais , Tamanho Corporal , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA de Cadeia Dupla/genética , Receptores de Esteroides/genética , Tetranychidae/crescimento & desenvolvimento
19.
PLoS One ; 14(7): e0219207, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339899

RESUMO

The distribution and diversity of RNA viruses in fungi is incompletely understood due to the often cryptic nature of mycoviral infections and the focused study of primarily pathogenic and/or economically important fungi. As most viruses that are known to infect fungi possess either single-stranded or double-stranded RNA genomes, transcriptomic data provides the opportunity to query for viruses in diverse fungal samples without any a priori knowledge of virus infection. Here we describe a systematic survey of all transcriptomic datasets from fungi belonging to the subphylum Pezizomycotina. Using a simple but effective computational pipeline that uses reads discarded during normal RNA-seq analyses, followed by identification of a viral RNA-dependent RNA polymerase (RdRP) motif in de novo assembled contigs, 59 viruses from 44 different fungi were identified. Among the viruses identified, 88% were determined to be new species and 68% are, to our knowledge, the first virus described from the fungal species. Comprehensive analyses of both nucleotide and inferred protein sequences characterize the phylogenetic relationships between these viruses and the known set of mycoviral sequences and support the classification of up to four new families and two new genera. Thus the results provide a deeper understanding of the scope of mycoviral diversity while also increasing the distribution of fungal hosts. Further, this study demonstrates the suitability of analyzing RNA-seq data to facilitate rapid discovery of new viruses.


Assuntos
Fungos/virologia , Genoma Viral , Transcriptoma/genética , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Fases de Leitura Aberta/genética , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética
20.
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
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