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1.
RNA ; 25(9): 1078-1090, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31164480

RESUMO

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.


Assuntos
Arabidopsis/genética , Indenos/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Interferência de RNA/efeitos dos fármacos , Tionas/farmacologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Argonauta/genética , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética
2.
Nucleic Acids Res ; 47(11): 5603-5616, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216042

RESUMO

The nematode Caenorhabditis elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both 'silencing' siRNAs bound by Worm-specific Argonautes (WAGO) and 'activating' siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here, we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partially rescued null mutant strain (WM193), this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements and an increased number of small RNAs that match enhancers in both drh-3 and csr-1 mutants. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to sites with increased silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.


Assuntos
Caenorhabditis elegans/genética , Cromatina/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Histonas/metabolismo , Metilação
3.
Methods Mol Biol ; 1974: 41-56, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31098994

RESUMO

In RNA interference (RNAi), silencing is achieved through the interaction of double-stranded small interfering RNAs (siRNAs) with essential RNAi pathway proteins, including Argonaute 2 (Ago2). Based on these interactions, one strand of the siRNA is loaded into Ago2 forming the active RNA-induced silencing complex (RISC). Optimal siRNAs maximize RISC activity against the intended target and minimize off-target silencing. To achieve the desired activity and specificity, selection of the appropriate siRNA strand for loading into Ago2 is essential. Here, we provide a protocol to quantify the relative loading of individual siRNA strands into Ago2, one factor in determining the capacity of a siRNA to achieve silencing activity and target specificity.


Assuntos
Proteínas Argonauta/genética , Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Carboxipeptidases/genética , Células HeLa , Humanos , Neoplasias/terapia , RNA de Cadeia Dupla/genética , Ribonuclease III/genética
4.
Folia Histochem Cytobiol ; 57(2): 56-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31112282

RESUMO

INTRODUCTION: Long interspersed nuclear elements-1 (L1), as the only one self-active retrotransposon of the mobile element, was found to be generally activated in tumor cells. The 5'UTR of L1 (L1-5'UTR) contains both sense and antisense bidirectional promoters, transcription products of which can generate double-strand RNA (dsRNA). In addition, L1-ORF1p, a dsRNA binding protein encoded by L1, is considered to engage in some RNA-protein (RNP) formation. Ago2, one of the RISC components, can bind to dsRNA to form RISC complex, but its role in L1 regulation still remains unclear. Due that the 5'UTR of L1 (L1-5'UTR) contains both sense and antisense bidirectional promoters, so the activities in both string were identified. A dsRNA-mediated regulation of L1-5'UTR, with the feedback regulation of L1-ORF1p as well as other key molecules engaged (Ago1-4) in this process, was also investigated. MATERIAL AND METHODS: Genomic DNA was extracted from HEK293 cells and subjected to L1-5'UTR prepa-ration by PCR. Report gene system pIRESneo with SV40 promoter was employed. The promoter activities of different regions in L1-5'UTR were identified by constructing these regions into pIRESneo, which SV40 region was removed prior, to generate different recombinant plasmids. The promoter activities in recombinant plasmids were detected by the luciferase expression assay. Western blot and co-immunoprecipitation were employed to identify proteins expression and protein-protein interaction respectively. RESULTS: Ago2 is a member of Agos family, which usually forms a RISC complex with si/miRNA and is involved in post- transcriptional regulation of many genes. Here L1-ORF1p and Ago2 conducts a regulation as a negative feedback for L1-5'UTR sense promoter. L1-ORF1p could form the immune complexes with Ago1, Ago2 and Ago4, respectively. CONCLUSIONS: L1-5'UTR harbors both sense and antisense promoter activity and a dsRNA-mediated regulation is responsible for L1-5'UTR regulation. Agos proteins and L1-ORF1p were engaged in this process.


Assuntos
Regiões 5' não Traduzidas , Proteínas Argonauta/genética , Regulação da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , RNA Interferente Pequeno/genética , Ribonucleoproteínas/genética , Sequência de Bases , Células HEK293 , Humanos , Mutação , Regiões Promotoras Genéticas
5.
RNA ; 25(9): 1098-1117, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31138671

RESUMO

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In Arabidopsis, this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action. Using AGO1:miRNA-mediated TR as a paradigm, we explored, with two unrelated polysome-isolation methods, which, among the ten Arabidopsis AGOs and numerous sRNA classes, interact with translation. We found that representatives of all three AGO-clades associate with polysomes, including the TGS-effector AGO4 and stereotypical 24-nt sRNAs that normally mediate TGS of transposons/repeats. Strikingly, approximately half of these annotated 24-nt siRNAs displayed unique matches in coding regions/introns of genes, and in pseudogenes, but not in transposons/repeats commonly found in their vicinity. Protein-coding gene-derived 24-nt sRNAs correlate with gene-body methylation. Those derived from pseudogenes belong to two main clusters defined by their parental-gene expression patterns, and are vastly enriched in AGO5, itself found on polysomes. Based on their tight expression pattern in developing and mature siliques, their biogenesis, and genomic/epigenomic features of their loci-of-origin, we discuss potential roles for these hitherto unknown polysome-enriched, pseudogene-derived siRNAs.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Argonauta/genética , Genes de Plantas/genética , Polirribossomos/genética , Pseudogenes/genética , RNA Interferente Pequeno/genética , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica/fisiologia , MicroRNAs/genética , Interferência de RNA/fisiologia , RNA de Plantas/genética
6.
Mol Cell ; 74(5): 982-995.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076285

RESUMO

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


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Mitocôndrias/genética , Fatores de Iniciação de Peptídeos/genética , RNA Helicases/genética , RNA Interferente Pequeno/genética , Animais , Drosophila melanogaster/genética , Endorribonucleases/genética , Feminino , Fertilidade/genética , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Mutação , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Proteínas de Ligação a RNA/genética
7.
BMC Bioinformatics ; 20(Suppl 4): 120, 2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999843

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. METHODS: We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3'UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples. RESULTS: For each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3'UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region. CONCLUSIONS: Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3'UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.


Assuntos
Proteínas Argonauta/metabolismo , Autoantígenos/metabolismo , Imunoprecipitação da Cromatina/métodos , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Argonauta/genética , Autoantígenos/genética , Sítios de Ligação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células MCF-7 , MicroRNAs/genética , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Máquina de Vetores de Suporte
8.
Mol Med Rep ; 19(5): 4468-4474, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942442

RESUMO

Increasing evidence has suggested that long non­coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has critical roles in multiple biological processes; however, few studies have reported on its function in heart disease. The present study indicated that NEAT1 expression is markedly downregulated in cardiomyocytes following ischemia/reperfusion injury in vivo and hydrogen peroxide treatment in vitro. Further experiments suggested that ectopic overexpression of NEAT1 suppresses cardiomyocyte apoptosis induced by hydrogen peroxide, as assessed by TUNEL assay and flow cytometry. In addition, using a dual­luciferase reporter assay, NEAT1 was demonstrated to directly interact with microRNA (miR)­125a­5p and overexpression of miR­125a­5p efficiently reversed the stimulatory effect of NEAT1 on B­cell lymphoma­2­like 12 (BCL2L12) expression. Furthermore, the results indicated that NEAT1 inhibits cardiomyocyte apoptosis via regulating the expression of BCL2L12, which appeared to be mediated via miR­125a­5p. In conclusion, the present study suggested that NEAT1 functions as a miR sponge to inhibit cardiomyocyte apoptosis and may be a novel therapeutic target for cardiomyocyte apoptosis­associated heart diseases.


Assuntos
Apoptose , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Antagomirs/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Regulação para Baixo/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Ratos , Ratos Sprague-Dawley
9.
RNA Biol ; 16(7): 950-959, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30982421

RESUMO

The large genome of the migratory locust (Locusta migratoria) genome accumulates massive amount of accumulated transposable elements (TEs), which show intrinsic transcriptional activities. Hampering the ability to precisely determine full-length RNA transcript sequences are exonized TEs, which produce numerous highly similar fragments that are difficult to resolve using short-read sequencing technology. Here, we applied a 5'-Cap capturing method using Nanopore long-read direct RNA sequencing to characterize full-length transcripts in their native RNA form and to analyze the TE exonization pattern in the locust transcriptome. Our results revealed the widespread establishment of TE exonization and a substantial contribution of TEs to RNA splicing in the locust transcriptome. The results of the transcriptomic spectrum influenced by Piwi expression indicated that TE-derived sequences were the main targets of Piwi-mediated repression. Furthermore, our study showed that Piwi expression regulates the length of RNA transcripts containing TE-derived sequences, creating an alternative UTR usage. Overall, our results reveal the transcriptomic characteristics of TE exonization in the species characterized by large and repetitive genomes.


Assuntos
Proteínas Argonauta/metabolismo , Elementos de DNA Transponíveis/genética , Éxons/genética , Gafanhotos/genética , Capuzes de RNA/metabolismo , Análise de Sequência de RNA , Animais , Proteínas Argonauta/genética , Sequência de Bases , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
10.
Nat Commun ; 10(1): 1626, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967537

RESUMO

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. So far, no comprehensive experimental annotation of functional miRNA target sites exists in Drosophila. Here, we generated a transcriptome-wide in vivo map of miRNA-mRNA interactions in Drosophila melanogaster, making use of single nucleotide resolution in Argonaute1 (AGO1) crosslinking and immunoprecipitation (CLIP) data. Absolute quantification of cellular miRNA levels presents the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two CLIP approaches, we identify a similar predictive potential to unambiguously assign thousands of miRNA-mRNA pairs from AGO1 interaction data at unprecedented depth, achieving higher signal-to-noise ratios than with computational methods alone. Quantitative RNA-seq and sub-codon resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites and their quantitative functional impact in Drosophila.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Animais , MicroRNAs/genética , MicroRNAs/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Análise de Sequência de RNA , Transcriptoma/genética
11.
Chem Commun (Camb) ; 55(35): 5139-5142, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30977478

RESUMO

The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.


Assuntos
Morfolinos/química , RNA Interferente Pequeno/química , Animais , Apolipoproteínas B/genética , Proteínas Argonauta/genética , Inativação Gênica , Humanos , Camundongos , Modelos Moleculares , Morfolinos/síntese química , Morfolinos/genética , Interferência de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética
12.
Nucleic Acids Res ; 47(7): 3594-3606, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30820541

RESUMO

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here, we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri, at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis. We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.


Assuntos
Proteínas Argonauta/genética , Evolução Molecular , Heligmosomatoidea/genética , RNA Interferente Pequeno/genética , Animais , Caenorhabditis elegans/genética , Heligmosomatoidea/patogenicidade , Humanos , Filogenia
13.
PLoS Genet ; 15(3): e1008036, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865625

RESUMO

Transposable elements (TEs) are dynamically expressed at high levels in multiple human tissues, but the function of TE-derived transcripts remains largely unknown. In this study, we identify numerous TE-derived microRNAs (miRNAs) by conducting Argonaute2 RNA immunoprecipitation followed by small RNA sequencing (AGO2 RIP-seq) on human brain tissue. Many of these miRNAs originated from LINE-2 (L2) elements, which entered the human genome around 100-300 million years ago. L2-miRNAs derived from the 3' end of the L2 consensus sequence and thus shared very similar sequences, indicating that L2-miRNAs could target transcripts with L2s in their 3'UTR. In line with this, many protein-coding genes carried fragments of L2-derived sequences in their 3'UTR: these sequences served as target sites for L2-miRNAs. L2-miRNAs and their targets were generally ubiquitously expressed at low levels in multiple human tissues, suggesting a role for this network in buffering transcriptional levels of housekeeping genes. In addition, we also found evidence that this network is perturbed in glioblastoma. In summary, our findings uncover a TE-based post-transcriptional network that shapes transcriptional regulation in human cells.


Assuntos
Elementos de DNA Transponíveis , Elementos Nucleotídeos Longos e Dispersos , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Sequência de Bases , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , MicroRNAs/metabolismo
14.
Gene ; 699: 54-61, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30858133

RESUMO

Epigenetic regulatory changes alter the gene regulation function of DNA repeat elements in cancer and consequently promote malignant phenotypes. Some short tandem repeat sequences, distributed throughout the human genome, can play a role as cis-regulatory elements of the genes. Distributions of tandem long (≥10) and short (<10) A-T repeats in the genome are different depending on gene functions. Long repeats are more commonly found in housekeeping genes and may regulate genes in harmonious fashion. Mononucleotide A-repeats around transcription start sites interact with Argonaute proteins (AGOs) to regulate gene expression. miRNA-bound AGO alterations in cancer have been reported; consequently, these changes would affect genes containing mononucleotide A- and T-repeats. Here, we showed an unprecedented hallmark of gene regulation in cancer. We evaluated the gene expression profiles reported in the Gene Expression Omnibus and found a high density of 13-27 A-T repeats in the up-regulated genes in malignancies derived from the bladder, cervix, head and neck, ovary, vulva, breast, colon, liver, lung, prostate, kidney, thyroid, adrenal gland, bone, blood cells, muscle and brain. Transfection of cell-penetrating protein tag AGO1 containing poly uracils (CPP-AGO1-polyUs) to the lung cancer cell lines altered gene regulation depending on the presence of long A-T repeats. CPP-AGO1-polyUs limited cell proliferation and the ability of a cancer cell to grow into a colony in lung cancer cell lines. In conclusion, long A-T repeats up-regulated many genes in cancer that can be targeted by AGO1 to change the expression of many genes and limited cancer growth.


Assuntos
Proteínas Argonauta/genética , Fatores de Iniciação em Eucariotos/genética , Repetições de Microssatélites/genética , Neoplasias/genética , Transcrição Genética/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , MicroRNAs/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sítio de Iniciação de Transcrição/fisiologia , Transcriptoma/genética , Regulação para Cima/genética
15.
Gene ; 701: 104-112, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30905810

RESUMO

PIWI family member piwil1, which associates with Piwi-interacting RNA (piRNA), is responsible in regulation of germ cell differentiation and maintenance of reproductive stem cells. In this study, we analyzed the piwil1 gene in Paralichthys olivaceus. Bioinformatics analysis and structure prediction showed that piwil1 had the conserved domains: PAZ domain and PIWI domain. Expression analysis during embryonic development implied that piwil1 gene was maternally inherited. The tissue distribution showed a sexually dimorphic gene expression pattern, with higher expression level in testis than ovary. In situ hybridization results demonstrated that piwil1 was predominantly distributed in oogonia, oocytes, sertoli cells and spermatocytes. A CpG island was predicted in the 5'-flanking region of piwil1 gene, and its methylation levels showed significant disparity between males and females, indicating that the sexually dimorphic expression of piwil1 gene might be regulated by methylation. Furthermore, we explored the distinct roles of human chorionic gonadotropin and 17α-methyltestosterone in regulating the expression of piwil1, and found that piwil1 was interacting with the HPG axis hormones. These results indicated that piwil1 might play a crucial role in gonadal development and gametogenesis in Paralichthys olivaceus.


Assuntos
Proteínas Argonauta/biossíntese , Proteínas de Peixes/biossíntese , Linguado/crescimento & desenvolvimento , Regulação da Expressão Gênica , Oogênese/fisiologia , Espermatogênese/fisiologia , Animais , Proteínas Argonauta/genética , Feminino , Proteínas de Peixes/genética , Linguado/genética , Masculino , Oócitos/citologia , Oócitos/metabolismo , Oogônios/citologia , Oogônios/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo
16.
Proc Natl Acad Sci U S A ; 116(15): 7549-7558, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30902896

RESUMO

Proteins of the ARGONAUTE (AGO) family function in the epigenetic regulation of gene expression. Although the rice (Oryza sativa) genome encodes 19 predicted AGO proteins, few of their functions have thus far been characterized. Here, we show that the AGO protein OsAGO2 regulates anther development in rice. OsAGO2 was highly expressed in anthers. Knockdown of OsAGO2 led to the overaccumulation of reactive oxygen species (ROS) and abnormal anther development, causing premature initiation of tapetal programmed cell death (PCD) and pollen abortion. The expression level of Hexokinase 1 (OsHXK1) increased significantly, and the methylation levels of its promoter decreased, in plants with knocked-down OsAGO2 expression. Overexpression of OsHXK1 also resulted in the overaccumulation of ROS, premature initiation of PCD, and pollen abortion. Moreover, knockdown of OsHXK1 restored pollen fertility in OsAGO2 knockdown plants. Chromatin immunoprecipitation assays demonstrated that OsAGO2 binds directly to the OsHXK1 promoter region, suggesting that OsHXK1 is a target gene of OsAGO2. These results indicate that OsHXK1 controls the appropriate production of ROS and the proper timing of tapetal PCD and is directly regulated by OsAGO2 through epigenetic regulation.


Assuntos
Apoptose , Proteínas Argonauta/metabolismo , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hexoquinase/biossíntese , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Argonauta/genética , Técnicas de Silenciamento de Genes , Hexoquinase/genética , Oryza/genética , Proteínas de Plantas/genética , Pólen/genética , Pólen/metabolismo , Elementos de Resposta
17.
RNA ; 25(5): 620-629, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770397

RESUMO

The small interfering RNAs (siRNA) or microRNAs (miRNA) incorporated into the RNA-induced silencing complex with the Argonaute (Ago) protein associates with target mRNAs through base-pairing, which leads to the cleavage or knockdown of the target mRNA. The seed region of the s(m)iRNA is crucial for target recognition. In this work, a molecular dynamic simulation was utilized to study the thermodynamics and kinetic properties of the third seed base binding to the target in the presence of the PIWI/MID domain of Ago. The results showed that in the presence of the PIWI/MID domain, the entropy and enthalpy changes for the association of the seed base with the target were smaller than those in the absence of protein. The binding affinity was increased due to the reduced entropy penalty, which resulted from the preorganization of the seed base into the A-helix form. In the presence of the protein, the association barrier resulting from the unfavorable entropy loss and the dissociation barrier coming from the destruction of hydrogen bonding and base-stacking interactions were lower than those in the absence of the protein. These results indicate that the seed region is crucial for fast recognition and association with the correct target.


Assuntos
Proteínas Argonauta/química , Fatores de Iniciação em Eucariotos/química , MicroRNAs/química , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Ligações de Hidrogênio , Cinética , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Termodinâmica
18.
Mol Cancer ; 18(1): 28, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782188

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been revealed to be implicated in the carcinogenesis and progression of HCC. However, the expressions, clinical significances, and roles of most lncRNAs in HCC are still unknown. METHODS: The expression of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in HCC tissues and cell lines was detected by qRT-PCR and fluorescence in situ hybridization. Immunoblotting, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MCM3AP-AS1 in HCC cell proliferation, cell cycle and apoptosis in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of HCC cells after MCM3AP-AS1 knockdown. The interactions among MCM3AP-AS1, miR-194-5p and FOXA1 were measured by RNA pull-down, RNA immunoprecipitation and luciferase reporter assay. RESULTS: We revealed a novel oncogenic lncRNA MCM3AP-AS1, which is overexpressed in HCC and positively correlated with large tumor size, high tumor grade, advanced tumor stage and poor prognosis of HCC patients. MCM3AP-AS1 knockdown suppressed HCC cell proliferation, colony formation and cell cycle progression, and induced apoptosis in vitro, and depletion of MCM3AP-AS1 inhibited tumor growth of HCC in vivo. Mechanistically, MCM3AP-AS1 directly bound to miR-194-5p and acted as competing endogenous RNA (ceRNA), and subsequently facilitated miR-194-5p's target gene forkhead box A1 (FOXA1) expression in HCC cells. Interestingly, FOXA1 restoration rescued MCM3AP-AS1 knockdown induced proliferation inhibition, G1 arrest and apoptosis of HCC cells. CONCLUSIONS: Our results recognized MCM3AP-AS1 as a novel oncogenic lncRNA, which indicated poor clinical outcomes in patients with HCC. MCM3AP-AS1 exerted an oncogenic role in HCC via targeting miR-194-5p and subsequently promoted FOXA1 expression. Our findings suggested that MCM3AP-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.


Assuntos
Acetiltransferases/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Acetiltransferases/metabolismo , Adulto , Idoso , Animais , Apoptose/genética , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Metástase Linfática , Masculino , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida
19.
Nat Commun ; 10(1): 844, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783097

RESUMO

Argonaute (AGO) proteins are core components of RNA interference (RNAi) but the mechanisms of their regulation, especially at the post-translational level, remain unclear. Among the ten AGOs in Arabidopsis, only AGO2 is induced by bacterial infection and is known to positively regulate immunity. Here we show that the N-terminal domain of AGO2 is enriched with arginine-glycine RG/GR repeats, which are methylated by protein arginine methyltransferase5 (PRMT5). Arginine methylation has dual functions in AGO2 regulation. Methylated arginine residues can promote AGO2 protein degradation and are also bound by Tudor-domain proteins (TSNs), which can degrade AGO2-associated small RNAs (sRNAs). PRMT5 is down-regulated during infection and the prmt5 mutant is more resistant to bacteria. We speculate that reduced PRMT5 expression during infection may lead to reduced arginine methylation of AGO2, resulting in accumulation of both AGO2 and, via reduced interaction with TSNs, accumulation of AGO2-associated sRNAs, to promote plant immunity. These results reveal that both the arginine methylation writer (PRMT5) and readers (TSNs) can regulate AGO2-mediated RNAi.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arginina/metabolismo , Proteínas Argonauta/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas Argonauta/genética , Metilação , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Estabilidade Proteica , Pseudomonas syringae/patogenicidade , RNA de Plantas/metabolismo
20.
Nat Commun ; 10(1): 828, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783109

RESUMO

PIWI-interacting RNAs (piRNAs) are at the center of a small RNA-based immune system that defends genomes against the deleterious action of mobile genetic elements (transposons). PiRNAs are highly variable in sequence with extensive targeting potential. Their diversity is restricted by their preference to start with a Uridine (U) at the 5' most position (1U-bias), a bias that remains poorly understood. Here we uncover that the 1U-bias of Piwi-piRNAs is established by consecutive discrimination against all nucleotides but U, first during piRNA biogenesis and then upon interaction with Piwi's specificity loop. Sequence preferences during piRNA processing also restrict U across the piRNA body with the potential to directly impact target recognition. Overall, the uncovered signatures could modulate specificity and efficacy of piRNA-mediated transposon restriction, and provide a substrate for purifying selection in the ongoing arms race between genomes and their mobile parasites.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , RNA Interferente Pequeno/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Mutação , Ovário/metabolismo , Domínios Proteicos , RNA Interferente Pequeno/genética , Uracila/metabolismo , Uridina/genética , Uridina/metabolismo
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