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1.
Cell Host Microbe ; 29(9): 1333-1335, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34499857

RESUMO

SARS-CoV-2 has mutually illuminated our collective knowledge and knowledge gaps, particularly in antiviral defense and therapeutic strategies. A recent study in Science (Poirier et al., 2021) uncovers an ancient antiviral mechanism that mammals utilize to suppress viruses, including SARS-CoV-2 and Zika virus, that could have broad implications for therapeutic strategies.


Assuntos
Proteínas Argonauta/metabolismo , COVID-19/prevenção & controle , Interferons/imunologia , Interferência de RNA/fisiologia , Ribonuclease III/metabolismo , Infecção por Zika virus/prevenção & controle , Animais , Linhagem Celular , Células HEK293 , Humanos , RNA Interferente Pequeno/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Replicação Viral , Zika virus/genética , Zika virus/imunologia
2.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445396

RESUMO

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Deleção de Sequência , Pareamento de Bases , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , RNA/química , Ribonuclease III/genética
4.
Science ; 373(6551): 231-236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244417

RESUMO

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Interferência de RNA , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células-Tronco/enzimologia , Células-Tronco/virologia , Processamento Alternativo , Animais , Encéfalo/enzimologia , Encéfalo/virologia , Linhagem Celular , RNA Helicases DEAD-box/química , Humanos , Imunidade Inata , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Organoides/enzimologia , Organoides/virologia , Infecções por Vírus de RNA/enzimologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/química , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Replicação Viral , Zika virus/genética , Zika virus/imunologia , Zika virus/fisiologia , Infecção por Zika virus/enzimologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
5.
Nat Commun ; 12(1): 4268, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257295

RESUMO

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.


Assuntos
Proteínas de Drosophila/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/genética , Ribonuclease III/metabolismo , Imagem Individual de Molécula/métodos , Animais , Drosophila , Proteínas de Drosophila/genética , Modelos Teóricos , RNA Helicases/genética , Interferência de RNA/fisiologia , Ribonuclease III/genética
6.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
7.
PLoS Pathog ; 17(5): e1009549, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33984068

RESUMO

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.


Assuntos
Infecções por Alphavirus/tratamento farmacológico , Antivirais/farmacologia , RNA Helicases DEAD-box/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Ribonuclease III/metabolismo , Vírus da Floresta de Semliki/efeitos dos fármacos , Replicação Viral , eIF-2 Quinase/metabolismo , Infecções por Alphavirus/metabolismo , Infecções por Alphavirus/patologia , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Interferon Tipo I/farmacologia , Ribonuclease III/genética , eIF-2 Quinase/genética
8.
J Med Chem ; 64(11): 7404-7421, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34038111

RESUMO

Imbalance miRNAs contribute to tumor formation; therefore, the development of small-molecule compounds that regulate miRNA biogenesis is an important strategy in oncotherapy. Here, (-)-Gomisin M1 (GM) was found to modulate miRNA biogenesis to inhibit the proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells. GM modulated expression profiles of miRNA and protein in HCC cells and suppressed tumor growth in a mouse model. Mechanistically, GM affected miRNA maturation by targeting TAR RNA-binding protein 2 (TRBP), with an efficacy higher than that of enoxacin, and promoted the binding of TRBP with Dicer. Structural simplification and a preliminary structure-activity relationship study via the synthesis of 20 GM derivatives showed that compound 9 exhibited more potent inhibitory activity in HCC cell proliferation and affinity for TRBP than did GM. These results suggest that TRBP may be a novel potential therapeutic target in HCC and compound 9 may be a potential drug candidate for the treatment of HCC.


Assuntos
Compostos Policíclicos/química , Proteínas de Ligação a RNA/metabolismo , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Enoxacino/química , Enoxacino/metabolismo , Enoxacino/farmacologia , Enoxacino/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Compostos Policíclicos/metabolismo , Compostos Policíclicos/farmacologia , Compostos Policíclicos/uso terapêutico , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Ribonuclease III/química , Ribonuclease III/metabolismo , Relação Estrutura-Atividade , Transcriptoma/efeitos dos fármacos , Transplante Heterólogo
9.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33827953

RESUMO

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.


Assuntos
Antivirais/farmacologia , Crinivirus/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ipomoea batatas/virologia , Doenças das Plantas/virologia , Ribonuclease III/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Antivirais/química , Antivirais/metabolismo , Crinivirus/enzimologia , Crinivirus/fisiologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ensaios de Triagem em Larga Escala , Simulação de Acoplamento Molecular , Fotossíntese/efeitos dos fármacos , Interferência de RNA , Ribonuclease III/química , Ribonuclease III/metabolismo , Bibliotecas de Moléculas Pequenas/química , Proteínas Virais/antagonistas & inibidores
10.
RNA ; 27(6): 694-709, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33795480

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that function as critical posttranscriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small noncoding RNA. Here, we develop the target-oriented miRNA discovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs from Argonaute crosslinking and sequencing of hybrids (Ago-CLASH) data sets. Using this technique, we discovered a novel miRNA derived from a primate specific noncoding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independently of Drosha processing but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently up-regulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A's role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor noncoding RNA.


Assuntos
Biologia Computacional/métodos , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Regiões 3' não Traduzidas , Movimento Celular , RNA Helicases DEAD-box/metabolismo , Células HCT116 , Células HEK293 , Humanos , Carioferinas/metabolismo , Células MCF-7 , Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Nucleosídeo NM23 Difosfato Quinases/genética , Neoplasias/patologia , RNA Longo não Codificante/metabolismo , Ribonuclease III/metabolismo
11.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923780

RESUMO

DNA methylation maintains genome stability and regulates gene expression in plants. RNA-directed DNA methylation (RdDM) is critical for appropriate methylation. However, no efficient tools are available for the investigation of the functions of specific DNA methylation. In this study, the cucumber mosaic virus vector was used for targeted DNA methylation. Methylation was rapidly induced but gradually decreased from the 3' end of the target endogenous sequence in Nicotiana benthamiana, suggesting a mechanism to protect against the ectopic introduction of DNA methylation. Increasing 24-nt siRNAs blocked this reduction in methylation by down-regulating DCL2 and DCL4. RdDM relies on the sequence identity between RNA and genomic DNA; however, this identity does not appear to be the sole determinant for efficient DNA methylation. The current findings provide new insight into the regulation of DNA methylation and promote additional effort to develop efficient targeted DNA methylation in plants.


Assuntos
Cucumovirus/genética , Metilação de DNA , Genes de Plantas , Tabaco/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vetores Genéticos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
12.
Nucleic Acids Res ; 49(8): 4456-4471, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823555

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses miRNAs during latency. However, regulation of viral miRNAs remains largely unknown. Our prior studies demonstrated that MCPIP1 regulates KSHV miRNA biogenesis by degrading most KSHV pre-miRNAs through its RNase activity. Some viral pre-miRNAs are partially resistant to degradation by MCPIP1. Here, we further characterized MCPIP1 substrate specificity and its antiviral potential against KSHV infection. In vitro cleavage assays and binding assays showed that MCPIP1 cleavage efficiency is related to binding affinity. Motif-based sequence analysis identified that KSHV pre-miRNAs that are well degraded by MCPIP1 have a 5-base motif (M5 base motif) within their terminal loops and this motif region consists of multiple pyrimidine-purine-pyrimidine (YRY) motifs. We further demonstrated that mutation of this M5 base motif within terminal loop of pre-miRNAs inhibited MCPIP1-mediated RNA degradation. We also revealed that MCPIP1 has an antiviral effect against KSHV infection. MCPIP1 can reduce the expression of Dicer, which in turn restricts KSHV infection. Conclusively, our findings demonstrated that MCPIP1 inhibited KSHV infection and suppressed viral miRNA biogenesis by directly degrading KSHV pre-miRNAs and altering the expression of miRNA biogenesis factors.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Humanos , MicroRNAs/genética , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade de RNA/genética , RNA Viral/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo
13.
Nucleic Acids Res ; 49(9): 5294-5307, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33877360

RESUMO

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Mucor/enzimologia , Ribonuclease III/química , Ribonuclease III/metabolismo , Evolução Molecular , Proteínas Fúngicas/genética , Modelos Moleculares , Mucorales/enzimologia , Mucorales/patogenicidade , Domínios Proteicos , RNA/metabolismo , Ribonuclease III/genética , Virulência
14.
Biochem Biophys Res Commun ; 556: 45-52, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33838501

RESUMO

Micro-RNA mediated suppression of mRNA translation represents a major regulatory mode of post-transcriptional gene expression. Recently, the nucleoporin Nup358 was shown to interact with AGO protein, a key component of miRNA-induced silencing complex (miRISC), and facilitate the coupling of miRISC with target mRNA. Previous results suggested that SUMO-interacting motifs (SIMs) present on Nup358 mediate interaction with AGO protein. Here we show that Nup358-SIM has multiple interacting regions on AGO2, specifically within the N, PAZ and MID domains, with an affinity comparable to SIM-SUMO1 interaction. The study also unraveled specific residues involved in the interaction of AGO2 with miRNA-loading components such as Dicer and HSP90. Collectively, the results support the conclusion that multiple SIMs contribute to the association of Nup358 with AGO2.


Assuntos
Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Motivos de Aminoácidos , Proteínas Argonauta/genética , Sítios de Ligação , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Ligação Proteica , Domínios Proteicos , Ribonuclease III/metabolismo , Deleção de Sequência , Ressonância de Plasmônio de Superfície
15.
Cell Mol Life Sci ; 78(7): 3709-3724, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33733306

RESUMO

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.


Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Quadruplex G , MicroRNAs/metabolismo , RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , RNA Helicases DEAD-box/metabolismo , DNA/química , DNA/genética , Inibidores Enzimáticos/química , Humanos , MicroRNAs/genética , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease III/metabolismo
16.
Mol Plant ; 14(4): 647-663, 2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524550

RESUMO

The precise regulation of microRNA (miRNA) biogenesis is crucial for plant development, which requires core microprocessors and many fine tuners to coordinate their miRNA processing activity/specificity in fluctuating cellular environments. During de-etiolation, light triggers a dramatic accumulation of core microprocessors and primary miRNAs (pri-miRNAs) but decreases pri-miRNA processing activity, resulting in relatively constant miRNA levels. The mechanisms underlying these seemingly contradictory regulatory changes remain unclear. In this study, we identified forkhead-associated domain 2 (FHA2) as a light-stabilized suppressor of miRNA biogenesis. We found that FHA2 deficiency increased the level of mature miRNAs, accompanied by a reduction in pri-miRNAs and target mRNAs. Biochemical assays showed that FHA2 associates with the core microprocessors DCL1, HYL1, and SE, forming a complex to suppress their pri-miRNA processing activity. Further analyses revealed that FHA2 promotes HYL1 binding but inhibits the binding of DCL1-PAZ-RNase-RNA-binding domains (DCL1-PRR) to miRNAs, whereas FHA2 does not directly bind to these RNAs. Interestingly, we found that FHA2 protein is unstable in the dark but stabilized by light during de-etiolation. Consistently, disruption of FHA led to defects in light-triggered changes in miRNA expression and reduced the survival rate of de-etiolated seedlings after prolonged light deprivation. Collectively, these data suggest that FHA2 is a novel light-stabilized suppressor of miRNA biogenesis and plays a role in fine-tuning miRNA processing during de-etiolation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Luz , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética
17.
Commun Biol ; 4(1): 207, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594200

RESUMO

Environmental conditions can cause phenotypic changes, part of which can be inherited by subsequent generations via soma-to-germline communication. However, the signaling molecules or pathways that mediate intertissue communication remain unclear. Here, we show that intertissue small RNA communication systems play a key role in the acquisition and inheritance of hormesis effects - stress-induced stress resistance - in Caenorhabditis elegans. The miRNA-processing enzyme DRSH-1 is involved in both the acquisition and the inheritance of hormesis, whereas worm-specific Argonaute (WAGO) proteins, which function with endo-siRNAs, are involved only in its inheritance. Further analyses demonstrate that the miRNA production system in the neuron and the small RNA transport machinery in the intestine are both essential for its acquisition and that both the transport of small RNAs in the germline and the germline Argonaute HRDE-1 complex are required for its inheritance. Our results thus demonstrate that overlapping and distinct roles of small RNA systems in the acquisition and inheritance of hormesis effects.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Hormese/genética , MicroRNAs/metabolismo , Transporte de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Hereditariedade , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Intestinos , MicroRNAs/genética , Neurônios/metabolismo , Pressão Osmótica , Estresse Oxidativo , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonuclease III/genética
18.
Microbiome ; 9(1): 57, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637135

RESUMO

BACKGROUND: Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. RESULTS: By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae, while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. CONCLUSION: Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota. Video abstract.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Metilação de DNA/genética , Microbiota , Raízes de Plantas/microbiologia , RNA de Plantas , Ribonuclease III/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Microbiota/genética , Raízes de Plantas/genética , RNA Ribossômico 16S/genética , Ribonuclease III/genética
19.
Theranostics ; 11(7): 3376-3391, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537093

RESUMO

Background: Colorectal cancer (CRC) and the associated metastatic lesions are reported to be hypoxic. Hypoxia is a common feature in the tumor microenvironment and a potent stimulant of CRC. We have identified a regulatory role of Nur77 on Akt activation to enhance ß-catenin signaling essential for CRC progression under hypoxic conditions. Methods: The functional role of Nur77 in hypoxia-induced EMT was examined by scattering assays to monitor the morphologies of CRC cell lines under 1% O2. Sphere formation assays were performed to investigate whether Nur77 induced cancer stem cell-like properties in hypoxic CRC cells. The expression of various epithelial-to-mesenchymal transition (EMT) and stemness markers was analyzed by qPCR and Western blotting. Finally, Nur77 function and signaling in vivo was ascertained in subcutaneous tumor xenograft or liver metastasis model in nude mice using CRC cells stably transfected with appropriate constructs. Results: Herein, we show, for the first time, that Nur77 is a novel regulator of microRNA biogenesis that may underlie its significant tumor-promoting activities in CRC cells under hypoxia. Mechanistically, Nur77 interacted with the tumor suppressor protein p63, leading to the inhibition of p63-dependent transcription of Dicer, an important miRNA processor and subsequent decrease in the biogenesis of let-7i-5p which targeted the 3'UTR of p110α mRNA and regulated its stability. Knockdown of Nur77 or overexpression of let-7i-5p inhibited the tumor metastasis in vivo. Conclusion: Our data uncovered a novel mechanistic link connecting Nur77, Akt, and invasive properties of CRC in the hypoxic microenvironment.


Assuntos
Adenocarcinoma/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/genética , RNA Helicases DEAD-box/genética , Hipóxia/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ribonuclease III/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , RNA Helicases DEAD-box/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia/metabolismo , Hipóxia/mortalidade , Hipóxia/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ribonuclease III/metabolismo , Transdução de Sinais , Análise de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Tumoral , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nucleic Acids Res ; 49(5): 2522-2536, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33561291

RESUMO

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.


Assuntos
Quadruplex G , MicroRNAs/química , MicroRNAs/metabolismo , Contração Miocárdica/genética , Miócitos Cardíacos/metabolismo , Animais , Benzilisoquinolinas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Quadruplex G/efeitos dos fármacos , Regulação da Expressão Gênica , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonuclease III/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
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