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1.
PLoS One ; 15(12): e0228233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347517

RESUMO

RNA interference (RNAi) plays key roles in post-transcriptional and chromatin modification levels as well as regulates various eukaryotic gene expressions which are involved in stress responses, development and maintenance of genome integrity during developmental stages. The whole mechanism of RNAi pathway is directly involved with the gene-silencing process by the interaction of Dicer-Like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) gene families and their regulatory elements. However, these RNAi gene families and their sub-cellular locations, functional pathways and regulatory components were not extensively investigated in the case of economically and nutritionally important fruit plant sweet orange (Citrus sinensis L.). Therefore, in silico characterization, gene diversity and regulatory factor analysis of RNA silencing genes in C. sinensis were conducted by using the integrated bioinformatics approaches. Genome-wide comparison analysis based on phylogenetic tree approach detected 4 CsDCL, 8 CsAGO and 4 CsRDR as RNAi candidate genes in C. sinensis corresponding to the RNAi genes of model plant Arabidopsis thaliana. The domain and motif composition and gene structure analyses for all three gene families exhibited almost homogeneity within the same group members. The Gene Ontology enrichment analysis clearly indicated that the predicted genes have direct involvement into the gene-silencing and other important pathways. The key regulatory transcription factors (TFs) MYB, Dof, ERF, NAC, MIKC_MADS, WRKY and bZIP were identified by their interaction network analysis with the predicted genes. The cis-acting regulatory elements associated with the predicted genes were detected as responsive to light, stress and hormone functions. Furthermore, the expressed sequence tag (EST) analysis showed that these RNAi candidate genes were highly expressed in fruit and leaves indicating their organ specific functions. Our genome-wide comparison and integrated bioinformatics analyses provided some necessary information about sweet orange RNA silencing components that would pave a ground for further investigation of functional mechanism of the predicted genes and their regulatory factors.


Assuntos
Citrus sinensis/genética , Regulação da Expressão Gênica de Plantas/genética , Interferência de RNA/fisiologia , Proteínas Argonauta/genética , Simulação por Computador , Etiquetas de Sequências Expressas , Frutas/metabolismo , Perfilação da Expressão Gênica/métodos , Genes de Plantas/genética , Genoma de Planta/genética , Família Multigênica/genética , Filogenia , Proteínas de Plantas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonuclease III/genética , Fatores de Transcrição/metabolismo
2.
Nat Commun ; 11(1): 5797, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199684

RESUMO

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.


Assuntos
Proteínas Argonauta/genética , Células Germinativas/metabolismo , Mutação/genética , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Interferência de RNA , Adolescente , Animais , Proteínas Argonauta/química , Criança , Pré-Escolar , Análise por Conglomerados , Dendritos/metabolismo , Fibroblastos/metabolismo , Inativação Gênica , Células HEK293 , Hipocampo/patologia , Humanos , Camundongos , Simulação de Dinâmica Molecular , Neurônios/metabolismo , Fosforilação , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Ratos , Transcriptoma/genética
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(10): 1373-1379, 2020 Oct 30.
Artigo em Chinês | MEDLINE | ID: mdl-33118502

RESUMO

OBJECTIVE: To analyze the correlation between the single nucleotide polymorphisms (SNPs) in the promoter of Piwil1 gene and gastric cancer. METHODS: The expression of Piwil1 mRNA in the tumor tissues of 3 patients with gastric cancer was detected by RT-qPCR, and RNA-Sequencing data from the Cancer RNA-Seq Nexus were analyzed for Piwil1 mRNA expression in gastric patients. Blood samples were collected from 24 gastric cancer patients and 29 healthy control subjects for PCR amplification of Piwil1 gene promoter region. The SNP loci in the promoter region of Piwil1 gene were determined by direct sequencing, and the results were analyzed by SnapGene software. RESULTS: Analysis of the data from Cancer RNA-Seq Nexus and the results of RT-qPCR in 3 gastric cancer patients all showed significantly increased Piwil1 expression in gastric cancer tissues compared with the adjacent tissues. Seven SNP loci in two CpG regions of the Piwil1 gene promoter were genotyped, and only one SNP locus was found to be related to gastric cancer. The frequencies of GG, GA, and AA genotypes at the rs28416520 locus in CpG 67 region were 79.2%, 16.7%, and 4.1% in the gastric cancer group, and were 37.9%, 55.2%, and 6.9% in the control group, respectively, showing a significantly higher frequency of the GG genotype in gastric cancer group (OR=0.144, 95%CI: 0.045-0.564, χ2=9.071, P < 0.01). The frequency of allele G of the rs28416520 locus was significantly higher in gastric cancer group than in the control group (87.5% vs 65.5%; OR=0.271, 95%CI: 0.099-0.766, χ2=6.856, P < 0.01). The genotype or allele frequencies of the other 6 SNPs locus did not differ significantly between gastric cancer group and control group. CONCLUSIONS: The expression of Piwil1 is increased in gastric cancer tissues as compared with the adjacent tissues. The GG genotype and G allele of rs28416520 within CpG 67 region are associated with an increased risk of gastric cancer.


Assuntos
Proteínas Argonauta/genética , Polimorfismo de Nucleotídeo Único , Neoplasias Gástricas , Estudos de Casos e Controles , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética
4.
Anticancer Res ; 40(10): 5539-5544, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988877

RESUMO

BACKGROUND/AIM: Endothelin-1 (ET-1) is overexpressed in many types of cancer, inhibiting the release of the microRNA 15a (miR-15a) and inducing the production of Mxi-2. Our aim was to identify a molecular complex regulating p53 activity in prostate cancer (PCa). MATERIALS AND METHODS: DU145 cells were treated with ET-1, MAPK p38 inhibitor, Endothelin A receptor inhibitor (ETAR inhibitor) and Endothelin B receptor inhibitor (ETBR inhibitor). Extracts were analysed using Western Blot, immunoprecipitation and qRT-PCR. Furthermore, prostate cancer patient samples were analysed using qRT-PCR and ELISA. RESULTS: The hypothesised molecular complex was identified, with miR-15a, microRNA 1285 (miR-1285) and Mxi-2 levels up-regulated in patients in relation to increasing aggressiveness of PCa. CONCLUSION: A complex composed of Argonaut 2 (Ago2)/Mxi-2/miR-1285 is involved in PCa. The expression of Mxi-2 correlates with increasing PCa aggressiveness and might be used as a non-invasive marker for the diagnosis and progression of PCa.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/genética , Neoplasias da Próstata/genética , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Argonauta/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antagonistas do Receptor de Endotelina A/farmacologia , Antagonistas do Receptor de Endotelina B/farmacologia , Humanos , Masculino , MicroRNAs/genética , Neoplasias da Próstata/patologia , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
5.
Pestic Biochem Physiol ; 170: 104700, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980067

RESUMO

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmß-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmß-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.


Assuntos
Locusta migratoria/genética , MicroRNAs/genética , Animais , Proteínas Argonauta/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética
6.
Mol Cell Biol ; 40(20)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32778571

RESUMO

Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.


Assuntos
Proteínas Argonauta/genética , RNA Helicases DEAD-box/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias/genética , Ribonuclease III/genética , Regiões 3' não Traduzidas/genética , Células A549 , Proteínas Argonauta/metabolismo , Sítios de Ligação/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(36): 22390-22401, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848063

RESUMO

Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, PIWI proteins, a subfamily of the PAZ-PIWI domain (PPD) protein family, have emerged as promising candidates for targeted cancer therapy. PPD proteins are essential for small noncoding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the PIWI subfamily partners with PIWI-interacting RNA (piRNA). Both PIWI proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, PIWI proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of PIWI proteins in cancer. Here we report that one of the four PIWI proteins in humans, PIWIL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out the PIWIL1 gene (PIWIL1-KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, few bona fide piRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PIWIL1 does not affect its oncogenic function. Thus, PIWIL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a piRNA-independent function of PIWIL1 in promoting gastric cancer.


Assuntos
Proteínas Argonauta , RNA Interferente Pequeno , Neoplasias Gástricas , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Estômago/química , Estômago/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
8.
Nucleic Acids Res ; 48(16): 9235-9249, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32810257

RESUMO

Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5' untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1-44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19-37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.


Assuntos
Instabilidade Genômica/genética , Hepatite C/genética , MicroRNAs/genética , Biossíntese de Proteínas , Regiões 5' não Traduzidas/genética , Proteínas Argonauta/genética , Genoma Viral/genética , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/virologia , Humanos , Sítios Internos de Entrada Ribossomal/genética , Estabilidade de RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Replicação Viral/genética
9.
Nat Commun ; 11(1): 4242, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843637

RESUMO

Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/genética , Sítios de Ligação , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Mutação , Ligação Proteica , Proteômica , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética
10.
Life Sci ; 257: 118089, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659369

RESUMO

AIM: Hepatitis B virus (HBV) is a major cause of a variety of liver diseases. Existing antiviral drugs cannot eradicate HBV from our body, and the main reason is unclear on the molecular mechanism of HBV replication. Flap endonuclease 1 (FEN1) can repair relaxed circular DNA (HBV rcDNA) to covalently closed circular DNA (HBV cccDNA) that promotes HBV DNA replication, while its specific regulatory detail remains unclear. In addition, miR-146a is close related to regulation in HBV replication. This study aims to explore whether miR-146a regulates HBV cccDNA formation through FEN1. MAIN METHODS: We investigated the expression of miR-146a, FEN1 and HBV copies in HBV stable replication cell line HepG2.2.15 and its parent cell line HepG2 transfected miR-146a and FEN1 plasmid by qRT-PCR and western blot, to identify the cooperation of Argonaute-2 (Ago2) and miR-146a by Ago2 siRNA and Ago2 RNA Binding Protein Immunoprecipitation (RIP). KEY FINDINGS: Compared with the control group, we found that the expression of miR-146a was significantly up-regulated in HepG2.2.15, and the expression of FEN1 and HBV copies were also significantly up-regulated. On contrary, the expression of target gene of miR-146a, interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor-6 (TRAF6), was significantly decreased in HepG2.2.15. With the use of Ago2 siRNA and then Ago2 RIP, we found that Ago2 performed as a carrier for miR-146a to promote HBV replication. SIGNIFICANCE: The results suggest a novel miR-146a â†’ FEN1 â†’ HBV DNA regulatory axis in HBV replication life. Ago2 cooperates with miR-146a to regulate the transcription and expression level of FEN1 protein through the downstream target gene IRAK1/TRAF6, and to promote HBV replication.


Assuntos
Proteínas Argonauta/genética , Vírus da Hepatite B/fisiologia , MicroRNAs/genética , Replicação Viral/genética , DNA Circular/genética , DNA Viral/genética , Endonucleases Flap/genética , Células Hep G2 , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética
11.
Nat Commun ; 11(1): 3739, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719317

RESUMO

The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Inativação Gênica , Animais , Proteínas Argonauta/genética , Feminino , Feto/citologia , Genoma , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Testículo/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(27): 15977-15988, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581127

RESUMO

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.


Assuntos
Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/metabolismo , MicroRNAs/efeitos dos fármacos , MicroRNAs/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Animais , Antagomirs/farmacologia , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Biomarcadores , Modelos Animais de Doenças , Epilepsia , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteômica , Ratos , Ratos Sprague-Dawley , Convulsões/genética , Análise de Sistemas , Regulação para Cima/efeitos dos fármacos
13.
Nucleic Acids Res ; 48(13): 7439-7453, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32501500

RESUMO

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3'-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3'-UTR. Our results suggest that assumptions about miRNA action should be re-examined.


Assuntos
Regiões 3' não Traduzidas , Proteínas Argonauta/metabolismo , Inativação Gênica , Proteínas Argonauta/química , Proteínas Argonauta/genética , Sítios de Ligação , Células HCT116 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
14.
Nat Commun ; 11(1): 3147, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561720

RESUMO

Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade ß-catenin. Disruption of ß-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and ß-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.


Assuntos
Proteínas Argonauta/metabolismo , Senescência Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Germinativas/metabolismo , Animais , Proteínas Argonauta/genética , Caderinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Ovário/citologia , Ovário/metabolismo , Retroelementos/genética , Transdução de Sinais , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Receptores Toll-Like/metabolismo , beta Catenina/metabolismo
15.
Nat Commun ; 11(1): 3115, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561756

RESUMO

Reproduction-specific small RNAs are vital regulators of germline development in animals and plants. MicroRNA2118 (miR2118) is conserved in plants and induces the production of phased small interfering RNAs (phasiRNAs). To reveal the biological functions of miR2118, we describe here rice mutants with large deletions of the miR2118 cluster. Our results demonstrate that the loss of miR2118 causes severe male and female sterility in rice, associated with marked morphological and developmental abnormalities in somatic anther wall cells. Small RNA profiling reveals that miR2118-dependent 21-nucleotide (nt) phasiRNAs in the anther wall are U-rich, distinct from the phasiRNAs in germ cells. Furthermore, the miR2118-dependent biogenesis of 21-nt phasiRNAs may involve the Argonaute proteins OsAGO1b/OsAGO1d, which are abundant in anther wall cell layers. Our study highlights the site-specific differences of phasiRNAs between somatic anther wall and germ cells, and demonstrates the significance of miR2118/U-phasiRNA functions in anther wall development and rice reproduction.


Assuntos
Flores/crescimento & desenvolvimento , MicroRNAs/metabolismo , Oryza/crescimento & desenvolvimento , RNA de Plantas/metabolismo , RNA Interferente Pequeno/biossíntese , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Mutação , Organogênese Vegetal/genética , Oryza/genética , Epiderme Vegetal/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas
16.
Nat Commun ; 11(1): 2765, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488030

RESUMO

MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3' ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3' binding are sufficient to trigger extensive miRNA 3' modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3' end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , MicroRNAs/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas Argonauta/genética , Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Técnicas de Inativação de Genes , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/genética , RNA Nucleotidiltransferases/genética
17.
Life Sci ; 255: 117845, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32470449

RESUMO

AIMS: Renal fibrosis is the typical manifestation of progressive kidney disease and causes a severe threat to human health. Surging evidence has illustrated that miRNA plays a core role in the genesis and development of kidney fibrosis. MiR-542-3p has been testified to function as a facilitator in hepatic stellate cell activation and fibrosis. The purpose of study is to investigate the potential of miR-542-3p in renal tubulointerstitial fibrosis. MATERIALS AND METHODS: In this study, to establish renal fibrosis model in vivo and in vitro, we first conducted unilateral ureteral obstruction (UUO) on rats and high glucose (HG) treatment on the HK-2 cells. Histological and western blot analyses were utilized for assessment of renal fibrosis model. Luciferase reporter assay was carried out to explore the regulatory mechanism underlying miR-542-3p in renal fibrosis. KEY FINDINGS: MiR-542-3p was found to be highly expressed in renal fibrosis. Functional experiments revealed that overexpression of miR-542-3p accelerated the deterioration of kidney fibrosis and inhibition of miR-542-3p led to the opposite result. Through the aid of bioinformatics tool, the speculated miR-542-3p binding sites were uncovered in the 3'UTR of argonaute RISC component 1 (AGO1). Mechanism study elucidated that AGO1 was a direct target of miR-542-3p. Lastly, our findings suggested that miR-542-3p played a promoting role in renal fibrosis via repression of AGO1. SIGNIFICANCE: We justified that miR-542-3p induced kidney fibrogenesis both in vivo and in vitro through targeting AGO1, unveiling that miR-542-3p might be a promising option for the treatment of patients with renal fibrosis.


Assuntos
Proteínas Argonauta/genética , Fatores de Iniciação em Eucariotos/genética , Nefropatias/patologia , Rim/patologia , MicroRNAs/genética , Animais , Sítios de Ligação , Linhagem Celular , Biologia Computacional , Modelos Animais de Doenças , Fibrose , Glucose/metabolismo , Humanos , Nefropatias/genética , Masculino , Ratos , Ratos Sprague-Dawley , Obstrução Ureteral/patologia
18.
Mol Cell ; 78(5): 862-875.e8, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32348780

RESUMO

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inativação Gênica/fisiologia , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , RNA Nuclear/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
19.
PLoS Genet ; 16(4): e1008765, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339167

RESUMO

microRNAs (miRNAs) are increasingly recognized as important regulators of many biological processes in mosquitoes, vectors of numerous devastating infectious diseases. Identification of bona fide targets remains the bottleneck for functional studies of miRNAs. In this study, we used CLEAR-CLIP assays to systematically analyze miRNA-mRNA interactions in adult female Anopheles gambiae mosquitoes. Thousands of miRNA-target pairs were captured after direct ligation of the miRNA and its cognate target transcript in endogenous Argonaute-miRNA-mRNA complexes. Using two interactions detected in this manner, miR-309-SIX4 and let-7-kr-h1, we demonstrated the reliability of this experimental approach in identifying in vivo gene regulation by miRNAs. The miRNA-mRNA interaction dataset provided an invaluable opportunity to decipher targeting rules of mosquito miRNAs. Enriched motifs in the diverse targets of each miRNA indicated that the majority of mosquito miRNAs rely on seed-based canonical target recognition, while noncanonical miRNA binding sites are widespread and often contain motifs complementary to the central or 3' ends of miRNAs. The time-lapse study of miRNA-target interactomes in adult female mosquitoes revealed dynamic miRNA regulation of gene expression in response to varying nutritional sources and physiological demands. Interestingly, some miRNAs exhibited flexibility to use distinct sequences at different stages for target recognition. Furthermore, many miRNA-mRNA interactions displayed stage-specific patterns, especially for those genes involved in metabolism, suggesting that miRNAs play critical roles in precise control of gene expression to cope with enormous physiological demands associated with egg production. The global mapping of miRNA-target interactions contributes to our understanding of miRNA targeting specificity in non-model organisms. It also provides a roadmap for additional studies focused on regulatory functions of miRNAs in Anopheles gambiae.


Assuntos
Anopheles/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Animais , Anopheles/fisiologia , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Estágios do Ciclo de Vida , MicroRNAs/genética , RNA Mensageiro/genética , Reprodução
20.
Nucleic Acids Res ; 48(9): 4681-4697, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32297952

RESUMO

The miRNA pathway has three segments-biogenesis, targeting and downstream regulatory effectors. We aimed to better understand their cellular control by exploring the miRNA-mRNA-targeting relationships. We first used human evolutionarily conserved sites. Strikingly, AGOs 1-3 are all among the top 14 mRNAs with the highest miRNA site counts, along with ANKRD52, the phosphatase regulatory subunit of the recently identified AGO phosphorylation cycle; and the AGO phosphorylation cycle mRNAs share much more than expected miRNA sites. The mRNAs for TNRC6, which acts with AGOs to channel miRNA-mediated regulatory actions onto specific mRNAs, are also heavily miRNA-targeted. In contrast, upstream miRNA biogenesis mRNAs are not, and neither are downstream regulatory effectors. In short, binding site enrichment in miRNA targeting machinery mRNAs, but neither upstream biogenesis nor downstream effector mRNAs, was observed, endowing a cellular capacity for intensive and specific feedback control of the targeting activity. The pattern was confirmed with experimentally determined miRNA-mRNA target relationships. Moreover, genetic experiments demonstrated cellular utilization of this capacity. Thus, we uncovered a capacity for intensive, and specific, feedback-regulation of miRNA targeting activity directly by miRNAs themselves, i.e. segment-specific feedback auto-regulation of miRNA pathway, complementing miRNAs pairing with transcription factors to form hybrid feedback-loop.


Assuntos
Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas Argonauta/genética , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Retroalimentação Fisiológica , Humanos , Fosforilação , RNA Mensageiro/química , RNA-Seq
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