RESUMO
Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins, which select the guide strand, often with a 5' end sequence bias. However, Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development, two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2, Dcl3, and Dcl5. Through in vitro cleavage assays, we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and 5' AGA, while Dcl3 has a sequence preference for 5' UNG. Dcl5, however, has cleavage preferences for 5' UAG and 3' CUAC/UN, which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus, we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.
Assuntos
Paramecium/genética , Proteínas de Protozoários/metabolismo , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis/genética , Paramecium/metabolismo , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Clivagem do RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ribonuclease III/classificação , Ribonuclease III/genética , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. VIDEO ABSTRACT.
Assuntos
DNA Concatenado , Paramecium tetraurellia/genética , Núcleo Celular/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Elementos de DNA Transponíveis , Exodesoxirribonucleases/metabolismo , Paramecium tetraurellia/citologia , Paramecium tetraurellia/metabolismo , RNA/genética , Transcrição GênicaRESUMO
R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.
Assuntos
Estruturas R-Loop , Ribonucleases , Humanos , Estruturas R-Loop/genética , Ribonucleases/genética , RNA/genética , DNA , Replicação do DNA , DNA Helicases/genética , Ribonuclease H/genética , Ribonuclease H/metabolismo , Instabilidade GenômicaRESUMO
In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner LoqsPB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer1â LoqsPB heterodimer. The Dicer-1 dsRBD and three LoqsPB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer1â LoqsPB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
Assuntos
Proteínas de Drosophila , MicroRNAs , Animais , Ribonuclease III/genética , Ribonuclease III/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Ligação a RNA/metabolismo , Drosophila/genética , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicerâ¢-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
Assuntos
MicroRNAs , Ribonuclease III , Camundongos , Animais , Ribonuclease III/metabolismo , Interferência de RNA , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Transporte/metabolismo , Mamíferos/metabolismoRESUMO
RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.
Assuntos
Proteína DEAD-box 58/metabolismo , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/metabolismo , Receptores Imunológicos/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Crioeletrônica , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/ultraestrutura , Epitopos , Evolução Molecular , Células HEK293 , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/ultraestrutura , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/genética , Receptores Imunológicos/ultraestrutura , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/ultraestrutura , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/ultraestruturaRESUMO
In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
Assuntos
RNA Helicases DEAD-box , Interferon Tipo I , NF-kappa B , Infecções por Vírus de RNA , Ribonuclease III , Animais , Humanos , NF-kappa B/genética , Interferência de RNA , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Ribonuclease III/química , Ribonuclease III/genética , Ribonuclease III/metabolismo , Infecções por Vírus de RNA/enzimologiaRESUMO
MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.
Assuntos
Proteínas Argonautas/genética , Eritropoese/genética , MicroRNAs/genética , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Interferência de RNA , Ribonuclease III/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.
Assuntos
MicroRNAs/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Camundongos , MicroRNAs/genética , Cultura Primária de Células , RNA Nucleotidiltransferases/metabolismo , Ribonuclease III/metabolismoRESUMO
In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.
Assuntos
Proteínas de Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerase Dependente de RNA/genética , Ribonuclease III/genética , Transcrição Gênica , Arabidopsis/genética , Proteínas Argonautas/genética , Metilação de DNA/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
Invertebrates mainly rely on sequence-specific RNA interference (RNAi) to resist viral infections. Increasing studies show that double-stranded RNA (dsRNA) can induce sequence-independent protection and that Dicer-2, the key RNAi player that cleaves long dsRNA into small interfering RNA (siRNA), is necessary for this protection. However, how this protection occurs remains unknown. Herein, we report that it is caused by adenosine triphosphate (ATP)-hydrolysis accompanying the dsRNA-cleavage. Dicer-2 helicase domain is ATP-dependent; therefore, the cleavage consumes ATP. ATP depletion activates adenosine monophosphate-activated protein kinase (Ampk) and induces nuclear localization of Fork head box O (FoxO), a key transcriptional factor for dsRNA-induced genes. siRNAs that do not require processing cannot activate the transcriptional response. This study reveals a unique nonspecific antiviral mechanism other than the specific RNAi in shrimp. This mechanism is functionally similar to, but mechanistically different from, the dsRNA-activated antiviral response in vertebrates and suggests an interesting evolution of innate antiviral immunity.
Assuntos
Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina , RNA de Cadeia Dupla , Ribonuclease III , Animais , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Ribonuclease III/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Imunidade Inata , Transcrição GênicaRESUMO
Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.
Assuntos
Vírus de RNA , Ribonuclease III , Ribonuclease III/metabolismo , Ribonuclease III/genética , Vírus de RNA/imunologia , Vírus de RNA/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Ascomicetos/virologia , Interferência de RNA , Replicação Viral/genética , RNA Viral/metabolismo , RNA Viral/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , RNA de Cadeia Dupla/metabolismoRESUMO
MicroRNAs (miRNAs) play vital roles in the regulation of gene expression, a process known as miRNA-induced gene silencing. The human genome codes for many miRNAs, and their biogenesis relies on a handful of genes, including DROSHA, DGCR8, DICER1, and AGO1/2. Germline pathogenic variants (GPVs) in these genes cause at least three distinct genetic syndromes, with clinical manifestations that range from hyperplastic/neoplastic entities to neurodevelopmental disorders (NDDs). Over the past decade, DICER1 GPVs have been shown to lead to tumor predisposition. Moreover, recent findings have provided insight into the clinical consequences arising from GPVs in DGCR8, AGO1, and AGO2. Here we provide a timely update with respect to how GPVs in miRNA biogenesis genes alter miRNA biology and ultimately lead to their clinical manifestations.
Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Genótipo , Genoma Humano , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismoRESUMO
Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.
Assuntos
Hipocampo/metabolismo , MicroRNAs/genética , Microglia/fisiologia , Neurônios/fisiologia , Ribonuclease III/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Reparo do DNA , Feminino , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Atividade Motora , Plasticidade Neuronal , Ribonuclease III/genéticaRESUMO
Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.
Assuntos
Interferência de RNA , RNA de Cadeia Dupla , RNA Interferente Pequeno , Ribonuclease III , Animais , Ribonuclease III/genética , Ribonuclease III/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/genética , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Feminino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.
Assuntos
Aedes , Flavivirus , Febre Amarela , Animais , Humanos , Interferência de RNA , Febre Amarela/genética , Flavivirus/genética , Antivirais , RNA Interferente Pequeno/genéticaRESUMO
The adipose tissue plays a crucial role in metabolism and physiology, affecting animal lifespan and susceptibility to disease. In this study, we present evidence that adipose Dicer1 (Dcr-1), a conserved type III endoribonuclease involved in miRNA processing, plays a crucial role in the regulation of metabolism, stress resistance, and longevity. Our results indicate that the expression of Dcr-1 in murine 3T3L1 adipocytes is responsive to changes in nutrient levels and is subject to tight regulation in the Drosophila fat body, analogous to human adipose and hepatic tissues, under various stress and physiological conditions such as starvation, oxidative stress, and aging. The specific depletion of Dcr-1 in the Drosophila fat body leads to changes in lipid metabolism, enhanced resistance to oxidative and nutritional stress, and is associated with a significant increase in lifespan. Moreover, we provide mechanistic evidence showing that the JNK-activated transcription factor FOXO binds to conserved DNA-binding sites in the dcr-1 promoter, directly repressing its expression in response to nutrient deprivation. Our findings emphasize the importance of FOXO in controlling nutrient responses in the fat body by suppressing Dcr-1 expression. This mechanism coupling nutrient status with miRNA biogenesis represents a novel and previously unappreciated function of the JNK-FOXO axis in physiological responses at the organismal level.
Assuntos
Proteínas de Drosophila , MicroRNAs , Animais , Humanos , Camundongos , Drosophila/metabolismo , Longevidade/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Estresse Oxidativo/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , RNA Helicases DEAD-box/metabolismoRESUMO
Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The slDCL2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.
Assuntos
MicroRNAs/metabolismo , Vírus de RNA/fisiologia , Ribonuclease III/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Sistemas CRISPR-Cas , Suscetibilidade a Doenças , Solanum lycopersicum/enzimologia , Mutação , Doenças das Plantas/virologia , Pequeno RNA não Traduzido/metabolismo , Ribonuclease III/genéticaRESUMO
Adenosine deaminases that act on RNA (ADARs) convert adenosines (A) to inosines (I) in stretches of dsRNA. The biological purpose of these editing events for the vast majority of ADAR substrates is largely unknown. In this issue of Genes & Development, Reich and colleagues (pp. 271-282) demonstrate that in Caenorhabditis elegans, A-to-I editing in double-stranded regions of protein-coding transcripts protects these RNAs from targeting by the RNAi pathway. Disruption of this safeguard through loss of ADAR activity coupled with enhanced RNAi results in developmental abnormalities and profound changes in gene expression that suggest aberrant induction of an antiviral response. Thus, editing of cellular dsRNA by ADAR helps prevent host RNA silencing and inadvertent antiviral activity.
Assuntos
Antivirais , Caenorhabditis elegans/genética , Animais , Edição de RNA , Interferência de RNA , RNA de Cadeia Dupla , RadarRESUMO
Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of Caenorhabditis elegans development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in adr-1;adr-2 mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in adr-1;adr-2 embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway (rrf-3 or ergo-1) in adr-1;adr-2 mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A)+ RNA-seq revealed EAG down-regulation and antiviral gene induction in adr-1;adr-2;rrf-3 embryos, and these expression changes were dependent on rde-1 and rde-4 Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs.