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1.
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
3.
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
4.
Elife ; 82019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31621580

RESUMO

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.


Assuntos
Aedes/virologia , Imunidade Inata , Vírus de RNA/crescimento & desenvolvimento , Vírus de RNA/imunologia , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , DNA Complementar/metabolismo , DNA Viral/metabolismo , Proteínas de Drosophila/metabolismo
5.
Nat Commun ; 10(1): 4705, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624244

RESUMO

DNA methylation, repressive histone marks, and PIWI-interacting RNA (piRNA) are essential for the control of retrotransposon silencing in the mammalian germline. However, it remains unknown how these repressive epigenetic pathways crosstalk to ensure retrotransposon silencing in the male germline. Here, we show that UHRF1 is responsible for retrotransposon silencing and cooperates with repressive epigenetic pathways in male germ cells. Conditional loss of UHRF1 in postnatal germ cells causes DNA hypomethylation, upregulation of retrotransposons, the activation of a DNA damage response, and switches in the global chromatin status, leading to complete male sterility. Furthermore, we show that UHRF1 interacts with PRMT5, an arginine methyltransferase, to regulate the repressive histone arginine modifications (H4R3me2s and H3R2me2s), and cooperates with the PIWI pathway during spermatogenesis. Collectively, UHRF1 regulates retrotransposon silencing in male germ cells and provides a molecular link between DNA methylation, histone modification, and the PIWI pathway in the germline.


Assuntos
Proteínas Argonauta/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Metilação de DNA , Proteína-Arginina N-Metiltransferases/genética , Retroelementos/genética , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas Argonauta/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Feminino , Inativação Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Espermatogênese/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Nat Commun ; 10(1): 4424, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562313

RESUMO

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5'-3' RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5'-3' exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Exorribonucleases/genética , Genes de Plantas , Mutagênese , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Estabilidade de RNA
7.
Plant Sci ; 288: 110218, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31521214

RESUMO

Salt stress is a major constraint to plant growth and development, and plants have developed sophisticated mechanisms to cope with it. AtAGO2, an argonaute protein, is known to play an important role in plant adaptation to salt stress; however, the molecular mechanism of this phenomenon remains essentially unexplored. Here, we performed the yeast two-hybrid assay and found an R3H-domain containing protein, designated as MUG13.4, interacting with AtAGO2. Further bimolecular fluorescence complement (BiFC), glutathione-S-transferase (GST) pull-down, and co-immunoprecipitation (Co-IP) assays confirmed that MUG13.4 interacted with AtAGO2, and MUG13.4 could affect the slicing activity of AtAGO2 associated with miR173. MUG13.4 and AtAGO2 were both predominantly expressed in seeds and roots. Phenotypic analyses of the single and double mutants under salt stress confirmed involvement of MUG13.4-AtAGO2 complex as a component of the salt tolerance mechanism. The mug13.4×ago2-1 double mutant displayed retarded growth and hypersensitivity to salt stress that was more pronounced than in mug13.4 or atago2-1 single mutants. TAS1c-tasiRNA generating system in Nicotiana benthamiana revealed that MUG13.4 could influence the slicing activity of AtAGO2. We also found that MUG13.4 increasingly changed the phenotype of slicer-defected mutants of AtAGO2 in response to salt stress. These findings suggested that the function of AtAGO2 upon salt stress was dependent on MUG13.4. Further investigation suggested that AtAGO2 improved Arabidopsis tolerance to salt stress by affecting operation of the SOS signaling cascade at the transcript level. Taken together, these findings reveal a novel function of MUG13.4 in adjusting Arabidopsis adaptation to salt stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas Argonauta/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Ligação a RNA/genética , Tolerância ao Sal/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Proteínas Argonauta/metabolismo , Perfilação da Expressão Gênica , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transdução de Sinais
8.
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
10.
Mol Cell ; 76(1): 44-56.e3, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31444105

RESUMO

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.


Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Evolução Molecular , Inosina/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Animais , Proteínas Argonauta/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Humanos , Magnésio/metabolismo , Manganês/metabolismo , Camundongos , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease H/química , Ribonuclease H/genética , Relação Estrutura-Atividade , Especificidade por Substrato
11.
BMC Genomics ; 20(1): 656, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31419938

RESUMO

BACKGROUND: Argonaute proteins (AGOs) are important players in the regulation of plant development by directing sRNAs to target mRNAs. In maize (Zea mays), AGO18b is a tassel-enriched and grass-specific AGO. Previous studies have shown that AGO18b is highly expressed in tassels during meiosis and negatively regulates determinacy of spikelet meristems. However, binding profile on RNAs and acting mechanisms of AGO18b remain unknown. RESULTS: In this study, we explored the binding profile of AGO18b in maize tassel by UV cross-linking RNA immunoprecipitation, followed by deep sequencing of these cDNA libraries (cRIP-seq), and systematically studied AGO18b-associated small RNAs and mRNAs by bioinformatics analysis. By globally analyzing the phased small-interfering RNA (phasiRNA) and miRNA abundance bound by AGO18b, we found AGO18b primarily binds to 21-nt phasiRNAs/miRNAs with a 5'-uridine and binds less strongly to 24-nt phasiRNAs with a 5'-adenosine in the premeiotic tassels. The abundance profile of AGO18b-associated miRNAs was different from their expression profile. Moreover, AGO18b strongly binds to miR166a-3p. We then obtained the AGO18b-bound mRNA targets of miR166a-3p by cRIP-seq, and confirmed the molecular function of AGO18b in regulating spikelet meristems. CONCLUSIONS: Our results indicated that AGO18b binds to phasiRNAs with obvious 5 prime end bias under different sRNA length. MiRNAs and their target mRNAs associated with AGO18b indicated the molecular mechanisms of AGO18b as a negative regulator of inflorescence meristem and tassel development through integrating both phasiRNAs and miRNA pathways, which extended our view of sRNA regulation in flower development and provided potential methods to control pollination in the future.


Assuntos
Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Proteínas de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Zea mays/genética , Biologia Computacional , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação , Inflorescência/crescimento & desenvolvimento , Inflorescência/metabolismo , Meiose , Meristema/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
12.
EMBO J ; 38(17): e102870, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31368590

RESUMO

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Feminino , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Genética
13.
EMBO J ; 38(13): e101153, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268608

RESUMO

microRNAs (miRNAs) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2-g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and supplementary (g13-g16) regions. Ago2 creates a "supplementary chamber" that accommodates up to five miRNA-target base pairs. Seed and supplementary chambers are adjacent to each other and can be bridged by an unstructured target loop of 1-15 nucleotides. Opening of the supplementary chamber may be constrained by tension in the miRNA 3' tail, as increases in miRNA length stabilize supplementary interactions. Contrary to previous reports, we demonstrate that optimal supplementary interactions can increase target affinity > 20-fold. These results provide a mechanism for extended miRNA targeting, suggest a function for 3' isomiRs in tuning miRNA targeting specificity, and indicate that supplementary interactions may contribute more to target recognition than is widely appreciated.


Assuntos
Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , MicroRNAs/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Mensageiro/química
14.
RNA ; 25(10): 1291-1297, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31289130

RESUMO

Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3'-UTRs of about 2000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3'-UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality.


Assuntos
Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Imunoprecipitação , Camundongos
15.
BMC Cancer ; 19(1): 712, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324173

RESUMO

BACKGROUND: Argonaute-2 (Ago2) is an essential component of microRNA biogenesis implicated in tumourigenesis. However Ago2 expression and localisation in breast cancer remains undetermined. The aim was to define Ago2 expression (mRNA and protein) and localisation in breast cancer, and investigate associations with clinicopathological details. METHODS: Ago2 protein was stained in breast cancer cell lines and tissue microarrays (TMAs), with intensity and localization assessed. Staining intensity was correlated with clinicopathological details. Using independent databases, Ago2 mRNA expression and gene alterations in breast cancer were investigated. RESULTS: In the breast cancer TMAs, 4 distinct staining intensities were observed (Negative, Weak, Moderate, Strong), with 64.2% of samples stained weak or negatively for Ago2 protein. An association was found between strong Ago2 staining and, the Her2 positive or basal subtypes, and between Ago2 intensity and receptor status (Estrogen or Progesterone). In tumours Ago2 mRNA expression correlated with reduced relapse free survival. Conversely, Ago2 mRNA was expressed significantly lower in SK-BR-3 (HER2 positive) and BT-20 (Basal/Triple negative) cell lines. Interestingly, high levels of Ago2 gene amplification (10-27%) were observed in breast cancer across multiple patient datasets. Importantly, knowledge of Ago2 expression improves predictions of breast cancer subtype by 20%, ER status by 15.7% and PR status by 17.5%. CONCLUSIONS: Quantification of Ago2 improves the stratification of breast cancer and suggests a differential role for Ago2 in breast cancer subtypes, based on levels and cellular localisation. Further investigation of the mechanisms affecting Ago2 dysregulation will reveal insights into the molecular differences underpinning breast cancer subtypes.


Assuntos
Proteínas Argonauta/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Argonauta/genética , Biomarcadores Tumorais/genética , Biópsia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Amplificação de Genes , Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/genética , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Estatísticas não Paramétricas
16.
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
17.
Nat Commun ; 10(1): 3389, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358756

RESUMO

Small RNAs have important functions. However, small RNAs in primate oocytes remain unexplored. Herein, we develop CAS-seq, a single-cell small RNA sequencing method, and profile the small RNAs in human oocytes and embryos. We discover a class of ~20-nt small RNAs that are predominantly expressed in human and monkey oocytes, but not in mouse oocytes. They are specifically associated with HIWI3 (PIWIL3), whereas significantly shorter than the commonly known PIWI-interacting RNAs (piRNAs), designated as oocyte short piRNAs (os-piRNAs). Notably, the os-piRNAs in human oocytes lack 2'-O-methylation at the 3' end, a hallmark of the classic piRNAs. In addition, the os-piRNAs have a strong 1U/10 A bias and are enriched on the antisense strands of recently evolved transposable elements (TEs), indicating the potential function of silencing TEs by cleavage. Therefore, our study has identified an oocyte-specific piRNA family with distinct features and provides valuable resources for studying small RNAs in primate oocytes.


Assuntos
Oócitos/metabolismo , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Elementos de DNA Transponíveis , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Oócitos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Testículo/citologia , Testículo/metabolismo
18.
Mol Cell ; 75(6): 1243-1255.e7, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31353209

RESUMO

MicroRNAs (miRNAs) broadly regulate gene expression through association with Argonaute (Ago), which also protects miRNAs from degradation. However, miRNA stability is known to vary and is regulated by poorly understood mechanisms. A major emerging process, termed target-directed miRNA degradation (TDMD), employs specialized target RNAs to selectively bind to miRNAs and induce their decay. Here, we report structures of human Ago2 (hAgo2) bound to miRNAs and TDMD-inducing targets. miRNA and target form a bipartite duplex with an unpaired flexible linker. hAgo2 cannot physically accommodate the RNA, causing the duplex to bend at the linker and display the miRNA 3' end for enzymatic attack. Altering 3' end display by changing linker flexibility, changing 3' end complementarity, or mutationally inducing 3' end release impacts TDMD efficiency, leading to production of distinct 3'-miRNA isoforms in cells. Our results uncover the mechanism driving TDMD and reveal 3' end display as a key determinant regulating miRNA activity via 3' remodeling and/or degradation.


Assuntos
Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Estabilidade de RNA , Animais , Proteínas Argonauta/genética , Células HEK293 , Humanos , MicroRNAs/genética , Células Sf9 , Spodoptera
19.
Mol Cell ; 75(4): 756-768.e7, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31350118

RESUMO

Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 105 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Drosophila/metabolismo , Homeostase/fisiologia , MicroRNAs/metabolismo , Análise de Sequência de RNA , Animais , Proteínas Argonauta/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , MicroRNAs/genética
20.
Genetics ; 213(1): 173-194, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31296532

RESUMO

MicroRNAs (miRNAs) associate with argonaute (AGO) proteins to post-transcriptionally modulate the expression of genes involved in various cellular processes. Herein, we show that loss of the Caenorhabditis elegans AGO gene alg-2 results in rapid and significantly increased germ cell apoptosis in response to DNA damage inflicted by ionizing radiation (IR). We demonstrate that the abnormal apoptosis phenotype in alg-2 mutant animals can be explained by reduced expression of mir-35 miRNA family members. We show that the increased apoptosis levels in IR-treated alg-2 or mir-35 family mutants depend on a transient hyperactivation of the C. elegans ERK1/2 MAPK ortholog MPK-1 in dying germ cells. Unexpectedly, MPK-1 phosphorylation occurs downstream of caspase activation and depends at least in part on a functional cell corpse-engulfment machinery. Therefore, we propose a refined mechanism, in which an initial proapoptotic stimulus by the core apoptotic machinery initiates the engulfment process, which in turn activates MAPK signaling to facilitate the demise of genomically compromised germ cells.


Assuntos
Apoptose , Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dano ao DNA , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Animais , Proteínas Argonauta/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo
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