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1.
Genomics ; 113(1 Pt 1): 331-343, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33321203

RESUMO

An outbreak, caused by an RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature, efficacy prediction and molecular docking analysis, 8 siRNA molecules were selected which are proposed to exert the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2.


Assuntos
/terapia , Química Computacional , Desenho de Fármacos , Terapia Genética/métodos , Simulação de Acoplamento Molecular , Interferência de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/química , RNA Viral/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Argonauta/química , Proteínas Argonauta/genética , Composição de Bases , /virologia , Evolução Molecular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Pandemias , Fosfoproteínas/genética , Filogenia , Dobramento de RNA , RNA Guia/química , RNA Guia/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , RNA Viral/genética , Alinhamento de Sequência , Termodinâmica
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.
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
4.
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
5.
J Nanobiotechnology ; 18(1): 65, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345308

RESUMO

Nanoparticle based gene delivery systems holds great promise. Superparamagnetic iron oxide nanoparticles (SPIONs) are being heavily investigated due to good biocompatibility and added diagnostic potential, rendering such nanoparticles theranostic. Yet, commonly used cationic coatings for efficient delivery of such anionic cargos, results in significant toxicity limiting translation of the technology to the clinic. Here, we describe a highly biocompatible, small and non-cationic SPION-based theranostic nanoparticles as novel gene therapy agents. We propose for the first-time, the usage of the microRNA machinery RISC complex component Argonaute 2 (AGO2) protein as a microRNA stabilizing agent and a delivery vehicle. In this study, AGO2 protein-conjugated, anti-HER2 antibody-linked and fluorophore-tagged SPION nanoparticles were developed (SP-AH nanoparticles) and used as a carrier for an autophagy inhibitory microRNA, MIR376B. These functionalized nanoparticles selectively delivered an effective amount of the microRNA into HER2-positive breast cancer cell lines in vitro and in a xenograft nude mice model of breast cancer in vivo, and successfully blocked autophagy. Furthermore, combination of the chemotherapy agent cisplatin with MIR376B-loaded SP-AH nanoparticles increased the efficacy of the anti-cancer treatment both in vitro in cells and in vivo in the nude mice. Therefore, we propose that AGO2 protein conjugated SPIONs are a new class of theranostic nanoparticles and can be efficiently used as innovative, non-cationic, non-toxic gene therapy tools for targeted therapy of cancer.


Assuntos
Proteínas Argonauta/química , Autofagia , Materiais Biocompatíveis/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Nanopartículas de Magnetita/química , MicroRNAs/metabolismo , Animais , Anticorpos/química , Anticorpos/imunologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cisplatino/química , Cisplatino/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Nus , MicroRNAs/química , Receptor ErbB-2/imunologia , Transplante Heterólogo
6.
PLoS Biol ; 18(3): e3000632, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163402

RESUMO

Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95°C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.


Assuntos
Proteínas de Drosophila/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/química , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dessecação , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Olho/patologia , Células HEK293 , Temperatura Alta , Humanos , Interações Hidrofóbicas e Hidrofílicas , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Longevidade , Masculino , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Estabilidade Proteica , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Solubilidade
7.
Nat Commun ; 11(1): 858, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051406

RESUMO

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila PIWI-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 Å resolution. A structural comparison of Piwi with other Argonautes highlights the PIWI-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons.


Assuntos
Proteínas Argonauta/classificação , Proteínas de Drosophila/química , Drosophila/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Bombyx/metabolismo , Linhagem Celular , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inativação Gênica , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , RNA não Traduzido
8.
Nucleic Acids Res ; 48(2): 862-878, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31740951

RESUMO

The Fragile X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation and is required for normal cognition. FMRP upregulates and downregulates the activity of microRNA (miRNA)-mediated silencing in the 3' UTR of a subset of mRNAs through its interaction with RNA helicase Moloney leukemia virus 10 (MOV10). This bi-functional role is modulated through RNA secondary structures known as G-Quadruplexes. We elucidated the mechanism of FMRP's role in suppressing Argonaute (AGO) family members' association with mRNAs by mapping the interacting domains of FMRP, MOV10 and AGO and then showed that the RGG box of FMRP protects a subset of co-bound mRNAs from AGO association. The N-terminus of MOV10 is required for this protection: its over-expression leads to increased levels of the endogenous proteins encoded by this co-bound subset of mRNAs. The N-terminus of MOV10 also leads to increased RGG box-dependent binding to the SC1 RNA G-Quadruplex and is required for outgrowth of neurites. Lastly, we showed that FMRP has a global role in miRNA-mediated translational regulation by recruiting AGO2 to a large subset of RNAs in mouse brain.


Assuntos
Proteínas Argonauta/genética , Proteína do X Frágil de Retardo Mental/genética , Biossíntese de Proteínas , RNA Helicases/genética , Animais , Proteínas Argonauta/química , Encéfalo/metabolismo , Proteína do X Frágil de Retardo Mental/química , Quadruplex G , Humanos , Camundongos , MicroRNAs/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Processamento de Proteína Pós-Traducional/genética , RNA Helicases/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
9.
J Chem Theory Comput ; 16(1): 688-699, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31751512

RESUMO

Argonaute (Ago) protein plays a central role in silencing gene expression by binding a "guide" strand to the base-pair with a complementary mRNA and degrading the mRNA. The current understanding of how Ago-guide and Ago-guide-mRNA complexes assemble is based mainly on static crystal structures; the associated kinetic pathways remain unknown/unclear. By simulating the successive binding of guide/target strand to Thermus thermophilus Ago (TtAgo) and computing the respective free energy landscapes, we directly visualize how TtAgo silencing complexes form and function. We show that the guide binding rate depends on its initial loading position onto TtAgo. Subsequent target recognition beyond the scissile 10-11 nucleotides must overcome a substantial energy barrier for TtAgo's nucleotide-binding groove to expand widely. This work reveals novel roles for the core TtAgo domains and shows how the kinetic barriers that must be overcome for critical structural changes to occur lead to target repression/cleavage.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , RNA Mensageiro/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonauta/química , Proteínas de Bactérias/química , Inativação Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , Termodinâmica , Thermus thermophilus/química , Thermus thermophilus/genética
10.
Talanta ; 208: 120486, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816728

RESUMO

Argonaute protein (AGO2) bound circulating cell-free miRNAs (ccf-miRs), in the recent years, has attracted great attention due to their differential abundance in biological fluids. In the present work, a selective and technically uncomplicated quantum dot (QD) nanoconjugate has been fabricated combining the specific affinity of the antibody and fluorescent property of QDs for the precise immuno-detection of AGO2-bound ccf-miRs in plasma samples. The electrophoretic mobility assay confirmed the conjugation of antibody with QDs. The detection methodology involves a highly specific antigen-antibody reaction between the AGO2 proteins of miRNA-induced silencing complex and the anti-AGO2 antibody conjugated with QDs. The recognition efficiency of QD-Ab nanoconjugates was analysed using flow cytometry and fluorometry. The flow cytometry results demonstrated a significant change in the fluorescence intensity of the prepared nanoconjugates upon capture of ccf-miRs in the plasma samples with respect to the samples devoid of any miRNAs. Fluorometry measurements exhibited corroboration with the flow cytometry results indicating the selectivity and reproducibility of the developed method. Current research highlights the translational significance of the methodology as a novel flow cytometry based immunoassay for detection of differentially expressed AGO2-bound miRNAs in clinical and field settings.


Assuntos
Anticorpos/química , Proteínas Argonauta/química , MicroRNAs/sangue , Nanoconjugados/química , Pontos Quânticos/química , Anticorpos/imunologia , Proteínas Argonauta/imunologia , Imunoensaio , MicroRNAs/química
11.
Mol Cells ; 42(12): 828-835, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31838836

RESUMO

PIWI Argonaute proteins and Piwi-interacting RNAs (piRNAs) are expressed in all animal species and play a critical role in cellular defense by inhibiting the activation of transposable elements in the germline. Recently, new evidence suggests that PIWI proteins and piRNAs also play important roles in various somatic tissues, including neurons. This review summarizes the neuronal functions of the PIWI-piRNA pathway in multiple animal species, including their involvement in axon regeneration, behavior, memory formation, and transgenerational epigenetic inheritance of adaptive memory. This review also discusses the consequences of dysregulation of neuronal PIWI-piRNA pathways in certain neurological disorders, including neurodevelopmental and neurodegenerative diseases. A full understanding of neuronal PIWI-piRNA pathways will ultimately provide novel insights into small RNA biology and could potentially provide precise targets for therapeutic applications.


Assuntos
Proteínas Argonauta/metabolismo , Neurônios/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Aprendizagem , Regeneração Nervosa , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Neurônios/metabolismo , RNA Interferente Pequeno/genética
12.
Science ; 366(6472)2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31806698

RESUMO

MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of messenger RNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA-target affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute-miRNA complexes and all sequences ≤12 nucleotides in length. This approach revealed noncanonical target sites specific to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks.


Assuntos
Proteínas Argonauta/química , MicroRNAs/química , Análise de Sequência de RNA/métodos , Sequência de Bases , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica
13.
Sci Rep ; 9(1): 16146, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31695077

RESUMO

Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Pareamento de Bases , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Hidrólise , Magnésio , Simulação de Acoplamento Molecular , Método de Monte Carlo , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Guia/química , RNA Guia/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/antagonistas & inibidores , RNA Viral/química , Complexo de Inativação Induzido por RNA , Relação Estrutura-Atividade , Termodinâmica
14.
Anal Chem ; 91(24): 15740-15747, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31714070

RESUMO

Rapid capture and identification of the intracellular target genes of microRNAs (miRNAs) are the key to understanding miRNA functions and development of RNA-based therapeutics. However, developing biochemical tools that can fish out the target genes of miRNAs in live cells is a significant technical challenge. Here, we report a remarkably simple yet powerful technology capable of loading virtually any miRNA into Ago2 of the RNA-induced silencing complexes (RISCs). This surprising discovery enables rapid capture and identification of target mRNAs and long noncoding RNAs. It is achieved by linking dibenzocyclooctyne (DBCO), a classical chemical moiety in copper-free click chemistry, to the 3' end of miRNAs. DBCO serves as a high-affinity tag to the Ago2 protein, thus boosting the formation of RISCs with miRNA target genes in living cells. Upon cell lysing, DBCO's routine function in click chemistry allows rapid enrichment of target genes for analysis without the need of additional molecular handles. A series of miR-21 and miR-27a target genes that were previously unknown were pulled down from various cell lines and identified with qRT-PCR, demonstrating the utility of this innovative technology in both transcriptomic research and RNA-based studies.


Assuntos
Proteínas Argonauta/metabolismo , Química Click/métodos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas Argonauta/química , Proteínas Argonauta/genética , Células HEK293 , Humanos , MicroRNAs/química , MicroRNAs/genética , RNA Mensageiro/química , RNA Mensageiro/genética
15.
Nat Cell Biol ; 21(10): 1261-1272, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570835

RESUMO

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
Biochem Biophys Res Commun ; 520(3): 514-519, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31610915

RESUMO

The recently discovered group of noncoding RNAs, which are fragments of tRNA molecules (tRFs), has not been fully characterized and its potential functions still require investigation. Porcine tRFs were characterized and compared to mouse and human tRFs. Two tRFs, 5' 32-33 nt and 3' 41-42 nt that are derived from the mature tRNAVal(CAC) and tRNAGly(GCC) were detected with the use of bioinformatics and the Northern blot method. The abundance of these tRFs in the case of Sus scrofa is restricted to the ovary and the kidney. The same tRFs were found in human cancer cells and in mouse sperm, circulating blood and its serum. The binding of selected sncRNAs (piRNA, 5'tRFVal(CAC) and miRNA) to the overexpressed PAZ domain of the PIWIL4 protein was also studied. It is noteworthy that porcine 5'tRFVal(CAC) and human 5'tRFVal(CAC)as well as 5'tRFGly(GCC) are bound to the PIWIL4 protein. The potential role of the analyzed tRFs in the development of mammals is also discussed.


Assuntos
Mamíferos/crescimento & desenvolvimento , Mamíferos/genética , RNA de Transferência/genética , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/genética , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Sequência Conservada , Evolução Molecular , Feminino , Humanos , Masculino , Mamíferos/metabolismo , Camundongos , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade da Espécie , Sus scrofa/metabolismo
17.
Nat Commun ; 10(1): 4390, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558728

RESUMO

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Proteínas Argonauta/química , Proteínas Argonauta/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Clostridium butyricum/genética , Clostridium butyricum/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Difusão , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Cinética , Microscopia de Fluorescência/métodos , Ligação Proteica , Estrutura Secundária de Proteína , RNA/química , RNA/genética , Imagem Individual de Molécula/métodos
18.
EMBO J ; 38(16): e100727, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31330067

RESUMO

Translational readthrough generates proteins with extended C-termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let-7a miRNA-binding site, and readthrough is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.


Assuntos
Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , MicroRNAs/genética , Biossíntese de Proteínas , Proteínas Argonauta/química , Autoantígenos/metabolismo , Sítios de Ligação , Códon de Terminação , Fatores de Iniciação em Eucariotos/química , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais
19.
Mol Cell ; 75(4): 741-755.e11, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31324449

RESUMO

Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.


Assuntos
Proteínas Argonauta/química , Modelos Químicos , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/química , Animais , Camundongos
20.
Mol Cell ; 75(4): 725-740.e6, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31324450

RESUMO

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9 Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.


Assuntos
Proteínas Argonauta/química , Fatores de Iniciação em Eucariotos/química , Proteínas de Ligação a RNA/química , Complexo de Inativação Induzido por RNA/química , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Estrutura Quaternária de Proteína , Células Sf9 , Spodoptera
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