Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.430
Filtrar
1.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805594

RESUMO

MicroRNAs applications were vastly studied throughout the years, spanning from potential cancer biomarkers to targeted therapies for various diseases. Out of these utilizations, this paper focuses on their role in male infertility. Approximately 10-15% of worldwide couples are affected by infertility. Out of these, 50% are due to male determinants. The majority of cases still have an undetermined cause. Previous studies have found that the aberrant expression of microRNAs could be linked to certain reproductive dysfunctions in males. Further on, this study looked into the most recent literature published on this subject in order to assess the connection between the up-/down-regulation of various microRNAs and the roles they play in male infertility. MicroRNAs were found to be abundant and stable in the seminal liquid, which led to a facile identification using regular RNA detection methods. It was observed that the concentration of microRNAs in semen was modified in the case of patients suffering from asthenozoospermia and azoospermia. Moreover, idiopathic male infertility was associated with a single nucleotide polymorphism of the microRNA binding site. Future studies should focus their attention on discovering future treatments against male infertility targeting specific microRNAs and also on developing new and improved contraceptive methods.


Assuntos
Proteínas Argonauta/genética , Astenozoospermia/genética , Azoospermia/genética , Infertilidade Masculina/genética , MicroRNAs/genética , Complexo de Inativação Induzido por RNA/genética , Adulto , Proteínas Argonauta/metabolismo , Astenozoospermia/metabolismo , Astenozoospermia/patologia , Azoospermia/metabolismo , Azoospermia/patologia , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , MicroRNAs/metabolismo , Polimorfismo de Nucleotídeo Único , Complexo de Inativação Induzido por RNA/metabolismo , Sêmen/citologia , Sêmen/metabolismo , Espermatogênese/genética
2.
Zoolog Sci ; 38(2): 103-111, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33812350

RESUMO

Nereidid polychaete Perinereis wilsoni is a homonomous metameric worm with a complete septum between each segment. Each segment has germ cells localized in the distal area of the parapodia. Perinereis wilsoni is also known to have high abilities of tissue regeneration; however, it is still unclear whether germ cells can regenerate in the healing tissue. To address this, we surgically operated the parapodia of an adult worm to remove germ cells from the segments and observed the germ cell regeneration using the germ cell genetic marker Pw-piwi. At day 20 post-surgical operation of the parapodia in one side of the segment, we found that Pw-piwi was expressed in the regenerating parapodia. We surgically operated the parapodia on both sides of the segment to remove the germ cells completely and it gave a similar result. However, before the expression of this gene marker in the regenerating parapodia, we observed that Pw-piwi was expressed in cells in the skin layer of the worm just after surgical operations. These Pw-piwi-positive cells were not observed in the un-operated worm. Our observations showed that germ cells of Perinereis wilsoni can regenerate even after the complete removal of germ cells from the defined habitat. The Pw-piwipositive cells that appeared in the skin layer after the disappearance of germ cells may be involved in the regeneration of new germ cells.


Assuntos
Proteínas Argonauta/metabolismo , Células Germinativas/metabolismo , Poliquetos/metabolismo , Animais , Proteínas Argonauta/genética , Regulação da Expressão Gênica , Filogenia , Ferimentos e Lesões
3.
Nat Commun ; 12(1): 2194, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850152

RESUMO

Multicellularity has coincided with the evolution of microRNAs (miRNAs), small regulatory RNAs that are integrated into cellular differentiation and homeostatic gene-regulatory networks. However, the regulatory mechanisms underpinning miRNA activity have remained largely obscured because of the precise, and thus difficult to access, cellular contexts under which they operate. To resolve these, we have generated a genome-wide map of active miRNAs in Caenorhabditis elegans by revealing cell-type-specific patterns of miRNAs loaded into Argonaute (AGO) silencing complexes. Epitope-labelled AGO proteins were selectively expressed and immunoprecipitated from three distinct tissue types and associated miRNAs sequenced. In addition to providing information on biological function, we define adaptable miRNA:AGO interactions with single-cell-type and AGO-specific resolution. We demonstrate spatial and temporal dynamicism, flexibility of miRNA loading, and suggest miRNA regulatory mechanisms via AGO selectivity in different tissues and during ageing. Additionally, we resolve widespread changes in AGO-regulated gene expression by analysing translatomes specifically in neurons.


Assuntos
Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Caenorhabditis elegans/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Sistema Nervoso , Isoformas de Proteínas
4.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673647

RESUMO

Small RNAs are essential to coordinate many cellular processes, including the regulation of gene expression patterns, the prevention of genomic instability, and the suppression of the mutagenic transposon activity. These processes determine the aging, longevity, and sensitivity of cells and an organism to stress factors (particularly, ionizing radiation). The biogenesis and activity of small RNAs are provided by proteins of the Argonaute family. These proteins participate in the processing of small RNA precursors and the formation of an RNA-induced silencing complex. However, the role of Argonaute proteins in regulating lifespan and radioresistance remains poorly explored. We studied the effect of knockdown of Argonaute genes (AGO1, AGO2, AGO3, piwi) in various tissues on the Drosophila melanogaster lifespan and survival after the γ-irradiation at a dose of 700 Gy. In most cases, these parameters are reduced or did not change significantly in flies with tissue-specific RNA interference. Surprisingly, piwi knockdown in both the fat body and the nervous system causes a lifespan increase. But changes in radioresistance depend on the tissue in which the gene was knocked out. In addition, analysis of changes in retrotransposon levels and expression of stress response genes allow us to determine associated molecular mechanisms.


Assuntos
Proteínas Argonauta/antagonistas & inibidores , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/crescimento & desenvolvimento , Longevidade/genética , RNA Interferente Pequeno/genética , Tolerância a Radiação/genética , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/efeitos da radiação , Feminino , Raios gama , Masculino , Especificidade de Órgãos , Interferência de RNA
5.
Mol Cell Biol ; 41(5)2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33685914

RESUMO

MicroRNAs (miRNAs) are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turnover in cancer cells, we explored the effect of target mRNAs on the abundance of the miRNAs that repress them. We have noted an accelerated extracellular vesicle (EV)-mediated export of miRNAs in presence of their target mRNAs in mammalian cells, and this target-driven miRNA-export process is retarded by Ago2-interacting protein GW182B. The GW182 group of proteins are localized to GW182 bodies or RNA processing bodies in mammalian cells, and GW182B-dependent retardation of miRNA export depends on GW body integrity and is independent of the HuR protein-mediated auxiliary pathway of miRNA export. Our data thus support the existence of a HuR-independent pathway of miRNA export in human cells that can be targeted in MDA-MB-231 cancer cells, to increase the level of cellular let-7a, a known negative regulator of cancer growth.


Assuntos
Proteínas Argonauta/genética , Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Argonauta/metabolismo , Autoantígenos/metabolismo , Humanos , MicroRNAs/genética , Neoplasias/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo
6.
Nat Plants ; 7(3): 342-352, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33633358

RESUMO

Plants use extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called 'cross-kingdom RNAi'. It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and annexins (ANNs), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not to non-EV-associated sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11 rh37 and ann1 ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RNA-binding proteins play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11 rh37 and ann1 ann2 showed increased susceptibility to Botrytis cinerea, suggesting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Vesículas Extracelulares/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Anexinas/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas Argonauta/metabolismo , Botrytis , RNA Helicases DEAD-box/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Proteoma , RNA Interferente Pequeno , Tetraspaninas/metabolismo
7.
Nucleic Acids Res ; 49(5): 2700-2720, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33590099

RESUMO

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.


Assuntos
Proteínas Argonauta/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/genética , Feminino , Genômica , Masculino , Mesocricetus , Metáfase , Fosforilação , RNA Interferente Pequeno/genética , Testículo/metabolismo
8.
Mol Cell ; 81(2): 223-225, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33482091

RESUMO

Han et al. (2020) and Shi et al. (2020) report that the E3 ubiquitin ligase ZSWIM8 senses when an RNA and an Argonaute protein-bound microRNA are extensively base paired and directs Argonaute destruction by the proteasome. The result is degradation of the microRNA.


Assuntos
MicroRNAs , Ubiquitina , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , MicroRNAs/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
RNA ; 27(4): 477-495, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33446492

RESUMO

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological processes such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration, suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterize planarian tRFs, we first annotated 457 tRNAs in S. mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians-the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of AGO1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalog of posttranscriptional regulatory systems in planaria, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.


Assuntos
Proteínas Argonauta/genética , Proteínas de Helminto/genética , Planárias/genética , RNA de Helmintos/genética , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Regeneração/genética , Algoritmos , Animais , Proteínas Argonauta/metabolismo , Pareamento de Bases , Sequência de Bases , Regulação da Expressão Gênica , Proteínas de Helminto/metabolismo , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Planárias/metabolismo , RNA de Helmintos/química , RNA de Helmintos/classificação , RNA de Helmintos/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/classificação , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/química , RNA de Transferência/classificação , RNA de Transferência/metabolismo
10.
Nucleic Acids Res ; 49(3): 1597-1608, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33444443

RESUMO

Argonaute (Ago) proteins are conserved nucleic acid-guided proteins present in all domains of life. Eukaryotic Argonaute proteins (eAgos) are key players in RNA interference pathways and function as RNA-guided RNA endonucleases at physiological temperatures. Although eAgos are considered to evolve from prokaryotic Argonaute proteins (pAgos), previously studied pAgos were unable to catalyze RNA-guided RNA cleavage at physiological temperatures. Here, we describe a distinctive pAgo from mesophilic bacteria Kurthia massiliensis (KmAgo). KmAgo utilizes DNA guides to cleave single-stranded DNA (ssDNA) and RNA targets with high activity. KmAgo also utilizes RNA guides to cleave ssDNA and RNA targets at moderate temperatures. We show that KmAgo can use 5' phosphorylated DNA guides as small as 9-mers to cut ssDNA and RNA, like Clostridium butyricum Ago. Small DNA binding confers remarkable thermostability on KmAgo, and we can suppress the guide-independent plasmid processing activity of empty KmAgo by elevating the DNA guide loaded temperature. Moreover, KmAgo performs programmable cleavage of double-stranded DNA and highly structured RNA at 37°C. Therefore, KmAgo can be regarded as a DNA-guided programmable omnipotent nuclease for cleaving most types of nucleic acids efficiently. This study broadens our understanding of Ago proteins and could expand the pAgo-based DNA and RNA manipulation toolbox.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , Planococáceas/enzimologia , RNA/metabolismo , Cátions Bivalentes , Quebras de DNA de Cadeia Dupla , Temperatura
11.
Methods Mol Biol ; 2167: 225-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712923

RESUMO

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3-5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.


Assuntos
Proteínas Argonauta/genética , Inativação Gênica , Infecções por HIV/genética , HIV-1/genética , Vírus Delta da Hepatite/genética , RNA Catalítico/genética , RNA Guia/genética , RNA Interferente Pequeno/genética , Proteínas Argonauta/metabolismo , Northern Blotting , Clonagem Molecular/métodos , Ensaios Enzimáticos/métodos , Vetores Genéticos , Células HEK293 , HIV-1/metabolismo , Humanos , Sequências Repetidas Invertidas/genética , Luciferases/metabolismo , Interferência de RNA , RNA Catalítico/metabolismo , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/virologia , Transfecção/métodos
12.
Biochem Biophys Res Commun ; 533(4): 1484-1489, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333714

RESUMO

In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas Argonauta/química , Proteínas Argonauta/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , DNA/química , DNA/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Ligação Proteica , RNA Guia/química , RNA Guia/genética , RNA Guia/metabolismo , Rhodobacter sphaeroides/genética
13.
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
14.
PLoS One ; 15(7): e0236710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735606

RESUMO

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the "hit" compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.


Assuntos
Proteínas Argonauta/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Espectroscopia de Ressonância Magnética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ressonância de Plasmônio de Superfície , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Simulação por Computador , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo
15.
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
16.
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
17.
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
18.
Nature ; 584(7822): 635-639, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32674113

RESUMO

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.


Assuntos
Metilação de DNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , DNA (Citosina-5-)-Metiltransferases/metabolismo , Elementos de DNA Transponíveis/genética , Feminino , Fertilidade/genética , Inativação Gênica , Genes de Partícula A Intracisternal/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , RNA Interferente Pequeno/biossíntese , Espermatogênese/genética
19.
Proc Natl Acad Sci U S A ; 117(30): 17764-17774, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661162

RESUMO

In cells, closely spaced microRNA (miRNA) target sites within a messenger RNA (mRNA) can act cooperatively, leading to more repression of the target mRNA than expected by independent action at each site. Using purified miRNA-Argonaute (AGO2) complexes, synthetic target RNAs, and a purified domain of TNRC6B (GW182 in flies) that is able to simultaneously bind multiple AGO proteins, we examined both the occupancies and binding affinities of miRNA-AGO2 complexes and target RNAs with either one site or two cooperatively spaced sites. On their own, miRNA-AGO2 complexes displayed little if any cooperative binding to dual sites. In contrast, in the presence of the AGO-binding region of TNRC6B, we observed strong cooperative binding to dual sites, with almost no singly bound target RNAs and substantially increased binding affinities and Hill coefficients. Cooperative binding was retained when the two sites were for two different miRNAs or when the two sites were bound to miRNAs loaded into two different AGO paralogs, AGO1 and AGO2. The improved binding affinity was attributable primarily to a reduced rate of dissociation between miRNA-AGO complexes and their dual-site targets. Thus, the multivalent binding of TNRC6 enables cooperative binding of miRNA-AGO complexes to target RNAs, thereby explaining the basis of cooperative action.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteínas Argonauta/metabolismo , Sítios de Ligação , Inativação Gênica , Humanos , Cinética , Modelos Biológicos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo
20.
Nat Struct Mol Biol ; 27(9): 790-801, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661421

RESUMO

Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by guiding Argonaute2 (AGO2) to its target site. Target site accessibility is critical for AGO2-target interactions, but how target site accessibility is controlled in vivo is poorly understood. Here, we use live-cell single-molecule imaging in human cells to determine rate constants of the AGO2 cleavage cycle in vivo. We find that the rate-limiting step in mRNA cleavage frequently involves unmasking of target sites by translating ribosomes. Target site masking is caused by heterogeneous intramolecular RNA-RNA interactions, which can conceal target sites for many minutes in the absence of translation. Our results uncover how dynamic changes in mRNA structure shape AGO2-target recognition, provide estimates of mRNA folding and unfolding rates in vivo, and provide experimental evidence for the role of mRNA structural dynamics in control of mRNA-protein interactions.


Assuntos
Proteínas Argonauta/metabolismo , RNA Mensageiro/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Clivagem do RNA , Dobramento de RNA , RNA Mensageiro/química , Ribossomos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...