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1.
Proc Natl Acad Sci U S A ; 118(31)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34330830

RESUMO

Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA-Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

2.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
3.
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
4.
Cell Rep ; 35(13): 109300, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34192539

RESUMO

The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, but the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates production of secondary small interfering RNAs (siRNAs) in plants. We show that the double-stranded RNA-binding protein SGS3 interacts directly with the 3' end of the microRNA in an Argonaute protein, resulting in ribosome stalling. Importantly, microRNA-mediated ribosome stalling correlates positively with efficient production of secondary siRNAs from target mRNAs. Our results illustrate a role of paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.

5.
EMBO Rep ; 22(7): e51342, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33973704

RESUMO

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposable elements and safeguard fertility in germ cells. Many protein factors required for piRNA biogenesis localize to perinuclear ribonucleoprotein (RNP) condensates named nuage, where target silencing and piRNA amplification are thought to occur. In mice, some of the piRNA factors are found in discrete cytoplasmic foci called processing bodies (P-bodies). However, the dynamics and biological significance of such compartmentalization of the piRNA pathway remain unclear. Here, by analyzing the subcellular localization of functional mutants of piRNA factors, we show that piRNA factors are actively compartmentalized into nuage and P-bodies in silkworm cells. Proper demixing of nuage and P-bodies requires target cleavage by the PIWI protein Siwi and ATP hydrolysis by the DEAD-box helicase BmVasa, disruption of which leads to promiscuous overproduction of piRNAs deriving from non-transposable elements. Our study highlights a role of dynamic subcellular compartmentalization in ensuring the fidelity of piRNA biogenesis.


Assuntos
Bombyx , Proteínas de Drosophila , Animais , Proteínas Argonauta/genética , Bombyx/genética , Bombyx/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Camundongos , RNA Interferente Pequeno/genética , Ribonucleoproteínas
6.
RNA ; 27(2): 151-162, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33177187

RESUMO

A key approach for improving siRNA efficacy is chemical modifications. Through an in silico screening of modifications at the 5'-end nucleobase of the guide strand, an adenine-derived compound called 6-(3-(2-carboxyethyl)phenyl)-purine (6-mCEPh-purine) was identified to improve the RNAi activity in cultured human cells and in vivo mouse models. Nevertheless, it remains unclear how this chemical modification enhances the siRNA potency. Here, we used a series of biochemical approaches to quantitatively evaluate the effect of the 6-mCEPh-purine modification at each step in the assembly of the RNAi effector complex called RISC. We found that the modification improves the formation of mature RISC at least in two different ways, by fixing the loading orientation of siRNA duplexes and increasing the stability of mature RISC after passenger strand ejection. Our data will provide a molecular platform for further development of chemically modified siRNA drugs.


Assuntos
Adenina/farmacologia , Proteínas Argonauta/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/agonistas , Complexo de Inativação Induzido por RNA/agonistas , Adenina/análogos & derivados , Adenina/síntese química , Proteínas Argonauta/metabolismo , Pareamento de Bases , Sequência de Bases , Células HEK293 , Humanos , Metilação , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo
7.
RNA ; 27(2): 163-173, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33177188

RESUMO

Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.


Assuntos
Adenina/farmacologia , Proteínas Argonauta/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/agonistas , Complexo de Inativação Induzido por RNA/agonistas , Adenina/análogos & derivados , Adenina/síntese química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Animais , Apolipoproteína B-100/antagonistas & inibidores , Apolipoproteína B-100/sangue , Apolipoproteína B-100/química , Apolipoproteína B-100/genética , Proteínas Argonauta/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Colesterol/sangue , Células HeLa , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Masculino , Metilação , Camundongos , Camundongos Knockout , Modelos Moleculares , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Uridina Monofosfato/química , Uridina Monofosfato/metabolismo
8.
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
9.
Nature ; 578(7794): 311-316, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996847

RESUMO

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.


Assuntos
Motivos de Aminoácidos , Sequência Consenso , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Fosfolipase D/química , Fosfolipase D/metabolismo , RNA Interferente Pequeno/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Sistema Livre de Células , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Metilação , Camundongos , RNA Helicases/metabolismo
10.
Autophagy ; 16(1): 190-192, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31718417

RESUMO

VCP (valosin containing protein) recognizes a wide variety of substrates and mediates their degradation via the ubiquitin-proteasome system and macroautophagy/autophagy. The substrate repertoire of VCP, however, is not fully understood. In our recent study, we found that Drosophila TER94/VCP mediates autophagic degradation of an Argonaute subfamily protein (AGO1), which binds microRNAs (miRNAs) and silences the expression of thousands of target genes. In the absence of TER94/VCP, miRNA-mediated gene silencing is globally impaired. Our findings reveal an unexpected connection between VCP and AGO, which may dramatically expand the biological significance of VCP.


Assuntos
Autofagia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/metabolismo , Animais , Proteínas Argonauta/genética , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo
11.
Cell Rep ; 28(5): 1144-1153.e4, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31365860

RESUMO

The Argonaute subfamily of proteins (AGO) loads microRNAs (miRNAs) to form the effector complex that mediates target gene silencing. Empty AGO, but not miRNA-loaded AGO, is selectively degraded across species. We have reported that the degradation of empty AGO is part of a quality control pathway that eliminates dysfunctional AGO. However, how empty AGO is degraded remains unclear. Here we show that the empty state of Drosophila Ago1 is degraded by autophagy. Comprehensive LC-MS/MS analyses, together with manipulation of the Ago1 ubiquitination level, revealed that VCP, which mediates selective autophagy, recognizes empty Ago1 via the Ufd1-Npl4 heterodimer. Depletion of VCP-Ufd1-Npl4 machinery impairs degradation of empty Ago1 and miRNA-mediated target gene silencing. Our findings reveal a direct link between empty AGO degradation and selective autophagy that ensures efficient miRNA function.


Assuntos
Proteínas Argonauta/metabolismo , Morte Celular Autofágica , Proteínas de Drosophila/metabolismo , Proteólise , Proteína com Valosina/metabolismo , Animais , Proteínas Argonauta/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína com Valosina/genética
12.
Mol Cell ; 73(1): 119-129.e5, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30503771

RESUMO

MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. We discovered a RING-type E3 ubiquitin ligase we named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. Our findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Lisina , MicroRNAs/genética , MicroRNAs/metabolismo , Ligação Proteica , Conformação Proteica , Proteólise , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
Mol Cell ; 70(4): 722-729.e4, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29775584

RESUMO

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form.


Assuntos
Proteínas Argonauta/química , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Conformação Proteica , Pequeno RNA não Traduzido/genética , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Ligação Proteica , Dobramento de Proteína , Interferência de RNA
14.
EMBO Rep ; 19(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29444933

RESUMO

PIWI-interacting RNAs (piRNAs) are germ cell-specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single-stranded RNAs to generate 5' termini of precursor piRNAs (pre-piRNAs) that are consecutively loaded into PIWI-family proteins. Subsequently, these pre-piRNAs are trimmed at their 3'-end by an exonuclease called Trimmer. Recently, poly(A)-specific ribonuclease-like domain-containing 1 (PNLDC1) was identified as the pre-piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post-natal pre-piRNAs. In addition, piRNA trimming defects in embryonic and post-natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post-meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1-mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.


Assuntos
Exorribonucleases/genética , Células Germinativas/crescimento & desenvolvimento , Meiose/genética , RNA Interferente Pequeno/genética , Ribonucleases/metabolismo , Animais , Inativação Gênica , Células Germinativas/metabolismo , Masculino , Camundongos , Proteínas Mitocondriais/genética , Mutação , Fosfolipase D/genética , Retroelementos/genética , Ribonucleases/genética , Espermatogênese/genética , Testículo/crescimento & desenvolvimento
15.
DNA Res ; 2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29360973

RESUMO

Bombyx mori macula-like virus (BmMLV) is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmMLV is currently characterized as an unclassified maculavirus. BmMLV accumulates at extremely high levels in cell lines derived from the silkworm, Bombyx mori, but it does not lead to lethality and establishes persistent infections. It is unknown how this insect maculavirus replicates and establishes persistent infections in insect cells. Here, we showed that BmMLV p15, which is located on a subgenomic fragment and is not found in plant maculaviruses, is highly expressed in BmMLV-infected silkworm cells and that p15 protein is required to establish BmMLV infections in silkworm cells. We also showed that two distinct small RNA-mediated pathways maintain BmMLV levels in BmMLV-infected silkworm cells, thereby allowing the virus to establish persistent infection. Virus-derived siRNAs and piRNAs were both produced as the infection progressed. Knockdown experiments demonstrated that the exogenous RNAi pathway alone or RNAi and piRNA pathways function cooperatively to silence BmMLV RNA and that both pathways are important for normal growth of BmMLV-infected silkworm cells. On the basis of our study, we propose a mechanism of how a plant virus-like insect virus can establish persistent infections in insect cells.

16.
Methods Mol Biol ; 1680: 131-143, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29030846

RESUMO

Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), silence protein expression from target mRNAs bearing their complementary sequences, via the formation of the effector complex called RNA-induced silencing complex (RISC). Although the mechanism of RISC assembly has been studied for nearly two decades, the detailed mechanism has still remained unclear in part due to the lack of a pure reconstitution system. Recently, we identified all the core proteins necessary for RISC assembly in flies and successfully recapitulated the assembly of catalytically active RISC with eight recombinant proteins. The reconstitution system provides a versatile framework for detailed studies of RISC assembly, including single molecule analysis as described in another chapter in this issue.


Assuntos
MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Proteínas Argonauta/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expressão Gênica , Inativação Gênica , Proteínas de Choque Térmico HSC70/metabolismo , Janus Quinases/metabolismo , Complexo de Inativação Induzido por RNA , Proteínas Recombinantes/metabolismo , Ribonuclease III/metabolismo , Fatores de Transcrição/metabolismo
17.
Methods Mol Biol ; 1680: 145-164, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29030847

RESUMO

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.


Assuntos
Imagem Molecular , Interferência de RNA , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Argonauta/metabolismo , Biologia Computacional/métodos , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , MicroRNAs/genética , Microscopia de Fluorescência , Imagem Molecular/métodos , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Software , Coloração e Rotulagem
18.
RNA ; 24(1): 6-11, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28971854

RESUMO

To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90ß, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90ß and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals.


Assuntos
Proteínas Argonauta/química , Complexo de Inativação Induzido por RNA/química , Trifosfato de Adenosina/química , Pareamento de Bases , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Humanos , Hidrólise , MicroRNAs/química , Multimerização Proteica , Termodinâmica
19.
Bio Protoc ; 8(1): e2673, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34179228

RESUMO

RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.

20.
Proc Natl Acad Sci U S A ; 114(47): 12483-12488, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29118143

RESUMO

The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.


Assuntos
Arginina/metabolismo , Proteínas Argonauta/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Cristalografia por Raios X , Epigênese Genética , Células HEK293 , Humanos , Masculino , Metilação , Modelos Moleculares , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Relação Estrutura-Atividade
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