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1.
Cell ; 139(1): 112-22, 2009 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-19804757

RESUMEN

MicroRNAs (miRNAs) govern an expanding number of biological and disease processes. Understanding the mechanisms by which the miRNA pathway is regulated, therefore, represents an important area of investigation. We determined that the human miRNA-generating complex is comprised of Dicer and phospho-TRBP isoforms. Phosphorylation of TRBP is mediated by the mitogen-activated protein kinase (MAPK) Erk. Expression of phospho-mimic TRBP and TRBP phosphorylation enhanced miRNA production by increasing stability of the miRNA-generating complex. Mitogenic signaling in response to serum and the tumor promoter PMA was dependent on TRBP phosphorylation. These effects were accompanied by a coordinated increase in levels of growth-promoting miRNA and reduced expression of let-7 tumor suppressor miRNA. Conversely, pharmacological inhibition of MAPK/Erk resulted in an anti-growth miRNA profile. Taken together, these studies indicate that the MAPK/Erk pathway regulates the miRNA machinery and suggest a general principle, wherein signaling systems target the miRNA pathway to achieve biological responses.


Asunto(s)
Sistema de Señalización de MAP Quinasas , MicroARNs/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo
2.
Mol Cell ; 41(5): 589-99, 2011 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-21362554

RESUMEN

The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3' overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5'-overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.


Asunto(s)
ARN Bicatenario/genética , Ribonucleasa III/química , Ribonucleasa III/metabolismo , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans , Drosophila , MicroARNs/metabolismo , Modelos Biológicos , Mutación , Estructura Terciaria de Proteína , ARN Bicatenario/química , ARN Interferente Pequeño/metabolismo
3.
Inorg Chem ; 54(4): 1627-33, 2015 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-25594823

RESUMEN

Here we demonstrate a novel and facile strategy of highly luminescent water-soluble Zn-doped AgIn5S8 (ZAIS) nanocrystals and ZAIS/ZnS core/shell structures, which were based on hydrothermal reaction between the acetate salts of the corresponding metals and sulfide precursor in the presence of l-cysteine at 110 °C in a Teflon-lined autoclave. The photoluminescent (PL) emission wavelength can be conveniently tuned from 560 to 650 nm by tailoring the stoichiometric ratio of [Ag]/[Zn]. The as prepared nanocrystals were characterized systematically and exhibit long PL lifetimes more than 100 ns. The influence of experimental conditions, including concentration of l-cysteine and reaction temperature, was investigated. In addition, we performed a coating procedure with the ZnS shell outside the ZAIS core and showed excellent PL quantum yields up to 35%. The in vitro experiment exhibited quite low cytotoxicity and marvelous biocompatibility, revealing their promising prospect in bioscience. Furthermore, the obtained ZAIS/ZnS nanocompounds (NCs) were covalently conjugated to alpha-fetoprotein antibodies and targeted fluorescent imaging for hepatocellular carcinoma cells was realized.


Asunto(s)
Carcinoma Hepatocelular/patología , Diagnóstico por Imagen , Colorantes Fluorescentes , Neoplasias Hepáticas/patología , Nanopartículas/química , Supervivencia Celular/efectos de los fármacos , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Células Hep G2 , Humanos , Indio/química , Plata/química , Solubilidad , Azufre/química , Temperatura , Agua/química , Zinc/química
4.
Cell Rep ; 43(3): 113882, 2024 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-38457341

RESUMEN

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein is a MT-associated protein (MAP) that directly binds MTs and stabilizes them by both promoting their growth and preventing their depolymerization. Furthermore, we demonstrate that A51R-MT interactions are conserved across A51R proteins from multiple poxvirus genera, and highly conserved, positively charged residues in A51R proteins mediate these interactions. Strikingly, we find that viruses encoding MT interaction-deficient A51R proteins fail to suppress a reactive oxygen species (ROS)-dependent antiviral response in macrophages that leads to a block in virion morphogenesis. Moreover, A51R-MT interactions are required for VV virulence in mice. Collectively, our data show that poxviral MAP-MT interactions overcome a cell-intrinsic antiviral ROS response in macrophages that would otherwise block virus morphogenesis and replication in animals.


Asunto(s)
Poxviridae , Replicación Viral , Animales , Ratones , Especies Reactivas de Oxígeno/metabolismo , Poxviridae/genética , Virus Vaccinia/fisiología , Proteínas Virales/metabolismo , Microtúbulos/metabolismo , Antivirales/metabolismo
5.
Phys Rev E ; 106(4-1): 044102, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-36397466

RESUMEN

We study the absorbing-state phase transition in the one-dimensional contact process under the combined influence of spatial and temporal random disorders. We focus on situations in which the spatial and temporal disorders decouple. Couched in the language of epidemic spreading, this means that some spatial regions are, at all times, more favorable than others for infections, and some time periods are more favorable than others independent of spatial location. We employ a generalized Harris criterion to discuss the stability of the directed percolation universality class against such disorder. We then perform large-scale Monte Carlo simulations to analyze the critical behavior in detail. We also discuss how the Griffiths singularities that accompany the nonequilibrium phase transition are affected by the simultaneous presence of both disorders.

6.
Protein Sci ; 29(6): 1429-1439, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32077153

RESUMEN

αß-tubulin subunits cycle through a series of different conformations in the polymer lattice during microtubule growing and shrinking. How these allosteric responses to different tubulin:tubulin contacts contribute to microtubule dynamics, and whether the contributions are evolutionarily conserved, remains poorly understood. Here, we sought to determine whether the microtubule-stabilizing effects (slower shrinking) of the ß:T238A mutation we previously observed using yeast αß-tubulin would generalize to mammalian microtubules. Using recombinant human microtubules as a model, we found that the mutation caused slow microtubule shrinking, indicating that this effect of the mutation is indeed conserved. However, unlike in yeast, ß:T238A human microtubules grew faster than wild-type and the mutation did not appear to attenuate the conformational change associated with guanosine 5'-triphosphate (GTP) hydrolysis in the lattice. We conclude that the assembly-dependent conformational change in αß-tubulin can contribute to determine the rates of microtubule growing as well as shrinking. Our results also suggest that an allosteric perturbation like the ß:T238A mutation can alter the behavior of terminal subunits without accompanying changes in the conformation of fully surrounded subunits in the body of the microtubule.


Asunto(s)
Microtúbulos/metabolismo , Mutación , Tubulina (Proteína)/genética , Regulación Alostérica , Humanos , Microtúbulos/química , Modelos Moleculares , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
7.
Elife ; 92020 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-32773040

RESUMEN

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αß-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αß heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/ultraestructura , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestructura , Microtúbulos/ultraestructura , Tubulina (Proteína)/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Células HeLa , Humanos , Conformación Proteica , Tirosina/química
8.
J Med Chem ; 63(22): 14054-14066, 2020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33180487

RESUMEN

A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as ß-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that ß-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with ß-tubulin harboring Cys239, but not ß3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in ß3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in ß-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.


Asunto(s)
Antineoplásicos/farmacología , Benzamidas/química , Colchicina/metabolismo , Microtúbulos/efectos de los fármacos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/química , Antineoplásicos/química , Sitios de Unión , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Microtúbulos/metabolismo , Conformación Proteica , Carcinoma Pulmonar de Células Pequeñas/patología , Relación Estructura-Actividad , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
9.
Methods Mol Biol ; 442: 11-27, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18369775

RESUMEN

RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA). In Drosophila melanogaster, the RNase III enzymes Dicer-1 and Dicer-2 generate miRNA and siRNA, respectively. We describe the methods for the expression, purification, and analysis of recombinant Dicer-1 and Dicer-2 enzymes. Our studies demonstrate that Dicer-1 and Dicer-2 display different substrate specificities and ATP requirements.


Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/enzimología , ARN Helicasas , Proteínas Recombinantes , Animales , Línea Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Humanos , ARN Helicasas/genética , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleasa III
10.
Mol Biol Cell ; 27(22): 3515-3525, 2016 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-27146111

RESUMEN

Microtubules are dynamic polymers of αß-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe-the switch from growing to shrinking-occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αß-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe.


Asunto(s)
Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Tamaño de la Célula , Simulación por Computador , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Microtúbulos/fisiología , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae
11.
J Mater Chem B ; 3(11): 2402-2410, 2015 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-32262071

RESUMEN

Cu doped Zn-In-S quantum dots (CZIS QDs) were synthesized by a hydrothermal method. The absorption and fluorescence peaks of CZIS QDs shifted monotonically to longer wavelengths with the increase of the Cu precursor and the decrease of Zn and In precursors. The dopant emission wavelength can be easily tuned in the whole visible region ranging from 465 nm to 700 nm by changing the molar ratio of Cu/Zn/In/S. On the basis of experimental results, it was testified that the emission of CZIS QDs was the trap state emission rather than the excitonic emission. The emission mechanisms of CZIS QDs were attributed to three kinds of approaches: (i) photogenerated holes efficiently move to trap states induced by Cu defects and recombine with the electrons in the energy level of sulfur vacancies; (ii) the holes in Cu trap states recombine with the electrons in the surface defect state; (iii) the electrons in the conduction band recombine with the holes in levels caused by Zn vacancies. After coating the ZnS shell around the CZIS core, the fluorescence quantum yield of CZIS QDs can reach 25-35%. CZIS/ZnS QDs conjugated with antibodies were successfully applied for labeling Hep-G2 liver cancer cells. The cytotoxicity studies revealed that the viabilities of the cells incubated with different concentrations of CZIS/ZnS QDs and at different times all remained at a high level of more than 90%. Hence, the CZIS/ZnS nanoparticle is a promising material as the fluorescent probe for biological applications.

12.
Elife ; 4: e10113, 2015 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-26439009

RESUMEN

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αß-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αß-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αß-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αß-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.


Asunto(s)
GTP Fosfohidrolasas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Regulación Alostérica , GTP Fosfohidrolasas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/genética
13.
FEMS Microbiol Lett ; 221(1): 53-7, 2003 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-12694910

RESUMEN

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.


Asunto(s)
Halobacterium salinarum/crecimiento & desarrollo , Plásmidos/genética , Proteínas Arqueales/genética , Composición de Base , Clonación Molecular , Medios de Cultivo , Replicación del ADN , Halobacterium salinarum/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Cloruro de Sodio
14.
Science ; 337(6096): 857-60, 2012 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-22904013

RESUMEN

Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αß-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αß-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αß-tubulin. TOG1 binds αß-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αß-tubulin that cannot be incorporated into microtubules, contacting α- and ß-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αß-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αß-tubulin and how they selectively recognize the growing end of the microtubule.


Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Microtúbulos/enzimología , Proteínas de Saccharomyces cerevisiae/química , Tubulina (Proteína)/química , Cristalografía por Rayos X , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Proteínas Asociadas a Microtúbulos/genética , Polimerizacion , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética
15.
Cell Metab ; 15(6): 895-904, 2012 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-22633452

RESUMEN

MicroRNAs (miRNAs) control gene expression by promoting degradation or repressing translation of target mRNAs. The components of the miRNA pathway are subject to diverse modifications that can modulate the abundance and function of miRNAs. Iron is essential for fundamental metabolic processes, and its homeostasis is tightly regulated. Here we identified iron chelators as a class of activator of the miRNA pathway that could promote the processing of miRNA precursors. We show that cytosolic iron could regulate the activity of the miRNA pathway through poly(C)-binding protein 2 (PCBP2). PCBP2 is associated with Dicer and promotes the processing of miRNA precursors. Cytosolic iron could modulate the association between PCBP2 and Dicer, as well as the multimerization of PCBP2 and its ability to bind to miRNA precursors, which can alter the processing of miRNA precursors. Our findings reveal a role of iron homeostasis in the regulation of miRNA biogenesis.


Asunto(s)
Homeostasis , Hierro/metabolismo , MicroARNs/metabolismo , Interferencia de ARN/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , 2,2'-Dipiridil/farmacología , Animales , ARN Helicasas DEAD-box/metabolismo , Deferoxamina/farmacología , Enoxacino/farmacología , Técnicas de Silenciamiento del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Quelantes del Hierro/farmacología , Ratones , Células 3T3 NIH , Multimerización de Proteína , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Ribonucleasa III/metabolismo
16.
Nat Struct Mol Biol ; 18(6): 650-7, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21552258

RESUMEN

Assembly of the RNA-induced silencing complex (RISC) consists of loading duplex (guide-passenger) siRNA onto Argonaute (Ago2) and removing the passenger strand. Ago2 contributes critically to RISC activation by nicking the passenger strand. Here we reconstituted duplex siRNA-initiated RISC activity using recombinant human Ago2 (hAgo2) and C3PO, indicating that C3PO has a critical role in hAgo2-RISC activation. Consistently, genetic depletion of C3PO compromised RNA silencing in mammalian cells. We determined the crystal structure of hC3PO, which reveals an asymmetric octamer barrel consisting of six translin and two TRAX subunits. This asymmetric assembly is critical for the function of C3PO as an endonuclease that cleaves RNA at the interior surface. The current work supports a Dicer-independent mechanism for human RISC activation, in which Ago2 directly binds duplex siRNA and nicks the passenger strand, and then C3PO activates RISC by degrading the Ago2-nicked passenger strand.


Asunto(s)
Carboxipeptidasas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Argonautas , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Endorribonucleasas/química , Humanos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína
17.
Nat Struct Mol Biol ; 17(1): 17-23, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19966796

RESUMEN

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.


Asunto(s)
Adenosina Trifosfato/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Interferencia de ARN , Complejo Silenciador Inducido por ARN/biosíntesis , Proteínas Argonautas , Western Blotting , Cartilla de ADN/genética , Factor 2 Eucariótico de Iniciación/aislamiento & purificación , Células HeLa , Humanos , Luciferasas , MicroARNs/metabolismo , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo
18.
Science ; 325(5941): 750-3, 2009 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-19661431

RESUMEN

The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage. We reconstituted long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Interferencia de ARN , Complejo Silenciador Inducido por ARN/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Argonautas , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Dominio Catalítico , Proteínas de Unión al ADN , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster/química , Drosophila melanogaster/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Bicatenario/química , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo
19.
Mech Dev ; 125(5-6): 475-85, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18299191

RESUMEN

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, we report here that r2d2(1) mutants have striking developmental defects. r2d2(1) mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. We show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2(1) mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. We conclude that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila.


Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/fisiología , Animales , Animales Modificados Genéticamente , Femenino , Masculino , MicroARNs/metabolismo , Modelos Genéticos , Mutación , Ovario/embriología , Ovario/metabolismo , Interferencia de ARN , Ribonucleasa III , Factores Sexuales , Transgenes
20.
J Biol Chem ; 282(39): 28373-28378, 2007 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-17666393

RESUMEN

In Drosophila melanogaster, the multidomain RNase III Dicer-1 (Dcr-1) functions in tandem with the double-stranded (ds)RNA-binding protein Loquacious (Loqs) to catalyze the maturation of microRNAs (miRNAs) from precursor (pre)-miRNAs. Here we dissect the molecular mechanism of pre-miRNA processing by the Dcr-1-Loqs complex. The tandem RNase III (RIII) domains of Dcr-1 form an intramolecular dimer such that one RIII domain cleaves the 3' strand, whereas the other cuts the 5' strand of pre-miRNA. We show that the functional core of Dcr-1 consists of a DUF283 domain, a PAZ domain, and two RIII domains. Dcr-1 preferentially associates with the Loqs-PB splice isoform. Loqs-PB uses the second dsRNA-binding domain to bind pre-miRNA and the third dsRNA-binding domain to interact with Dcr-1. Both domains of Loqs-PB are required for efficient miRNA production by enhancing the affinity of Dcr-1 for pre-miRNA. Thus, our results provide further insights into the functional anatomy of the Drosophila miRNA-generating enzyme.


Asunto(s)
Proteínas de Drosophila/metabolismo , MicroARNs/metabolismo , Complejos Multiproteicos/metabolismo , ARN Helicasas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Dimerización , Proteínas de Drosophila/genética , Drosophila melanogaster , Complejos Multiproteicos/genética , Unión Proteica/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/fisiología , ARN Helicasas/genética , Precursores del ARN/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo
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