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1.
Nature ; 607(7918): 393-398, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35768503

RESUMO

In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts1. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation2-5. Here we report cryo-electron microscopy structures of the Dicer-2-R2D2 and Dicer-2-R2D2-siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing.


Assuntos
Microscopia Crioeletrônica , Proteínas de Drosophila , RNA Helicases , RNA de Cadeia Dupla , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Ribonuclease III , Animais , Proteínas Argonautas/metabolismo , Pareamento de Bases , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , MicroRNAs/metabolismo , Multimerização Proteica , RNA Helicases/química , RNA Helicases/metabolismo , RNA Helicases/ultraestrutura , Interferência de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Ribonuclease III/ultraestrutura
2.
Nature ; 564(7736): E37, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30459470

RESUMO

In Fig. 1b of this Article, a U was inadvertently inserted after G15 in the D loop. The original Article has not been corrected.

3.
Nucleic Acids Res ; 50(22): 12997-13010, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36477368

RESUMO

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1-10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5' nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5' adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5' A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Metilação de DNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Nucleotídeos/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo
4.
Nucleic Acids Res ; 50(8): 4669-4684, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35380679

RESUMO

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient 'eudicot-type' DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5' phosphates and 3' overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.


Assuntos
Proteínas de Arabidopsis , Ribonuclease III , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Duplicação Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , RNA de Cadeia Dupla , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
5.
Proc Natl Acad Sci U S A ; 115(29): 7527-7532, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29967150

RESUMO

Throughout three domains of life, alanyl-tRNA synthetases (AlaRSs) recognize a G3:U70 base pair in the acceptor stem of tRNAAla as the major identity determinant of tRNAAla The crystal structure of the archaeon Archaeoglobus fulgidus AlaRS in complex with tRNAAla provided the basis for G3:U70 recognition with residues (Asp and Asn) that are conserved in the three domains [Naganuma M, et al. (2014) Nature 510:507-511]. The recognition mode is unprecedented, with specific accommodation of the dyad asymmetry of the G:U wobble pair and exclusion of the dyad symmetry of a Watson-Crick pair. With this conserved mode, specificity is based more on "fit" than on direct recognition of specific atomic groups. Here, we show that, in contrast to the archaeal complex, the Escherichia coli enzyme uses direct positive (energetically favorable) minor groove recognition of the unpaired 2-amino of G3 by Asp and repulsion of a competing base pair by Asn. Strikingly, mutations that disrupted positive recognition by the E. coli enzyme had little or no effect on G:U recognition by the human enzyme. Alternatively, Homo sapiens AlaRS selects G:U without positive recognition and uses Asp instead to repel a competitor. Thus, the widely conserved Asp-plus-Asn architecture of AlaRSs can select G:U in a straightforward (bacteria) or two different unconventional (eukarya/archaea) ways. The adoption of different modes for recognition of a widely conserved G:U pair in alanine tRNAs suggests an early and insistent role for G:U in the development of the genetic code.


Assuntos
Alanina-tRNA Ligase/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Modelos Moleculares , Motivos de Nucleotídeos , RNA de Transferência/química , Alanina-tRNA Ligase/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Mutação , RNA de Transferência/genética
6.
Nature ; 510(7506): 507-11, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24919148

RESUMO

Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNA(Ala) with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.


Assuntos
Alanina-tRNA Ligase/química , Archaeoglobus fulgidus/enzimologia , Archaeoglobus fulgidus/genética , Pareamento de Bases , RNA de Transferência de Alanina/química , RNA de Transferência de Alanina/genética , Aminoacilação de RNA de Transferência , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Especificidade por Substrato
7.
Nat Commun ; 14(1): 8341, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-38097570

RESUMO

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.


Assuntos
Genes Precoces , Proteína Quinase 1 Ativada por Mitógeno , Humanos , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Ativação Transcricional
8.
Proc Natl Acad Sci U S A ; 106(21): 8489-94, 2009 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-19423669

RESUMO

Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNA(Ala) by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AlaRS: AlaRS-DeltaC, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helix-loop-helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding.


Assuntos
Alanina-tRNA Ligase/química , Alanina-tRNA Ligase/metabolismo , Multimerização Proteica , Alanina-tRNA Ligase/genética , Aminoacilação , Archaeoglobus fulgidus/enzimologia , Archaeoglobus fulgidus/genética , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA de Transferência/química , RNA de Transferência/metabolismo
9.
Methods Mol Biol ; 2509: 209-231, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35796966

RESUMO

Single-molecule imaging is a powerful method for unveiling precise molecular mechanisms. Particularly, single-molecule analysis with total internal reflection fluorescence (TIRF ) microscopy has been successfully applied to the characterization of molecular mechanisms in ncRNA studies. Tracing interactions at the single-molecule level have elucidated the intermediate states of the reaction, which are hidden by ensemble averaging in combinational biochemical approaches, and clarified the key steps of the interaction. However, applying a single-molecule technique to ncRNA analysis still remains a challenge, requiring laborious trial and error to identify a suitable glass surface passivation method. In this chapter, we revisit the major glass surface passivation methods using polyethylene glycol (PEG) treatment and summarize a detailed protocol for single-molecule analysis of the dicing process of Dcr-2, which may apply piRNA studies in the future.


Assuntos
Vidro , Imagem Individual de Molécula , Vidro/química , Microscopia de Fluorescência/métodos , Nanotecnologia , Polietilenoglicóis/química , Imagem Individual de Molécula/métodos
10.
Nat Commun ; 12(1): 4268, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257295

RESUMO

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.


Assuntos
Proteínas de Drosophila/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/genética , Ribonuclease III/metabolismo , Imagem Individual de Molécula/métodos , Animais , Drosophila , Proteínas de Drosophila/genética , Modelos Teóricos , RNA Helicases/genética , Interferência de RNA/fisiologia , Ribonuclease III/genética
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