RESUMEN
Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is caused by mutations in the gene encoding the Fragile X messenger ribonucleoprotein (FMRP). FMRP is an evolutionarily conserved and neuronally enriched RNA-binding protein (RBP) with functions in RNA editing, RNA transport, and protein translation. Specific target RNAs play critical roles in neurodevelopment, including the regulation of neurite morphogenesis, synaptic plasticity, and cognitive function. The different biological functions of FMRP are modulated by its cooperative interaction with distinct sets of neuronal RNA and protein-binding partners. Here, we focus on interactions between FMRP and components of the microRNA (miRNA) pathway. Using the Drosophila S2 cell model system, we show that the Drosophila ortholog of FMRP (dFMRP) can repress translation when directly tethered to a reporter mRNA. This repression requires the activity of AGO1, GW182, and MOV10/Armitage, conserved proteins associated with the miRNA-containing RNA-induced silencing complex (miRISC). Additionally, we find that untagged dFMRP can interact with a short stem-loop sequence in the translational reporter, a prerequisite for repression by exogenous miR-958. Finally, we demonstrate that dFmr1 interacts genetically with GW182 to control neurite morphogenesis. These data suggest that dFMRP may recruit the miRISC to nearby miRNA binding sites and repress translation via its cooperative interactions with evolutionarily conserved components of the miRNA pathway.
Asunto(s)
Proteínas de Drosophila , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , MicroARNs , Neuritas , Biosíntesis de Proteínas , Animales , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , MicroARNs/genética , MicroARNs/metabolismo , Neuritas/metabolismo , Morfogénesis/genética , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Drosophila/metabolismo , Drosophila/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Unión ProteicaRESUMEN
Small noncoding RNAs (sncRNAs) regulate biological processes by impacting post-transcriptional gene expression through repressing the translation and levels of targeted transcripts. Despite the clear biological importance of sncRNAs, approaches to unambiguously define genome-wide sncRNA:target RNA interactions remain challenging and not widely adopted. We present CIMERA-seq, a robust strategy incorporating covalent ligation of sncRNAs to their target RNAs within the RNA-induced silencing complex (RISC) and direct detection of in vivo interactions by sequencing of the resulting chimeric RNAs. Modifications are incorporated to increase the capacity for processing low-abundance samples and permit cell-type-selective profiling of sncRNA:target RNA interactions, as demonstrated in mouse brain cortex. CIMERA-seq represents a cohesive and optimized method for unambiguously characterizing the in vivo network of sncRNA:target RNA interactions in numerous biological contexts and even subcellular fractions. Genome-wide and cell-type-selective CIMERA-seq enhances researchers' ability to study gene regulation by sncRNAs in diverse model systems and tissue types.
Asunto(s)
ARN Pequeño no Traducido , Análisis de Secuencia de ARN , Animales , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Ratones , Análisis de Secuencia de ARN/métodos , Humanos , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Genoma/genéticaRESUMEN
Essential amino acids (EAA) and microRNAs (miRs) control biological activity of a cell. Whether EAA regulates the activity of miR has never been demonstrated. Here, as proof-of-concept, a tryptophan (Trp, an EAA) complex containing Argonaute 2 (Ago2) and miRs including miR-193a (Trp/Ago2/miR-193a) is identified. Trp binds miR-193a-3p and interacts with Ago2. Trp/Ago2/miR-193a increases miR-193a-3p activity via enhancing Argonaute 2 (Ago2) RNase activity. Other miRs including miR-103 and miR-107 in the Trp complex enhance miR-193a activity by targeting the same genes. Mechanistically, the Trp/Ago2/miR-193a complex interacts with Trp-binding pockets of the PIWI domain of Ago2 to enhance Ago2 mediated miR activity. This newly formed Ago2/Trp/miR-193a-3p complex is more efficient than miR-193a-3p alone in inhibiting the expression of targeted genes and inhibiting colon cancer liver metastasis. The findings show that Trp regulates miR activity through communication with the RNA-induced silencing complexes (RISC), which provides the basis for tryptophan based miR therapy.
Asunto(s)
Proteínas Argonautas , Neoplasias del Colon , Neoplasias Hepáticas , MicroARNs , Complejo Silenciador Inducido por ARN , Triptófano , Triptófano/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Humanos , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Neoplasias del Colon/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Ratones , Animales , Línea Celular Tumoral , Modelos Animales de EnfermedadRESUMEN
During infection, adenoviruses inhibit the cellular RNA interference (RNAi) machinery by saturating the RNA-induced silencing complex (RISC) of the host cells with large amounts of virus-derived microRNAs (mivaRNAs) that bind to the key component of the complex, Argonaute 2 (AGO2). In the present study, we investigated AGO2 as a prominent player at the intersection between human adenovirus 5 (HAdV-5) and host cells because of its ability to interfere with the HAdV-5 life cycle. First, the ectopic expression of AGO2 had a detrimental effect on the ability of the virus to replicate. In addition, in silico and in vitro analyses suggested that endogenous microRNAs (miRNAs), particularly hsa-miR-7-5p, have similar effects. This miRNA was found to be able to target the HAdV-5 DNA polymerase mRNA. The inhibitory effect became more pronounced upon overexpression of AGO2, likely due to elevated AGO2 levels, which abolished the competition between cellular miRNAs and mivaRNAs for RISC incorporation. Collectively, our data suggest that endogenous miRNAs would be capable of significantly inhibiting viral replication if adenoviruses had not developed a mechanism to counteract this function. Eventually, AGO2 overexpression-mediated relief of the RISC-saturating action of mivaRNAs strongly enhanced the effectiveness of artificial miRNAs (amiRNAs) directed against the HAdV-5 preterminal protein (pTP) mRNA, suggesting a substantial benefit of co-expressing amiRNAs and AGO2 in RNAi-based strategies for the therapeutic inhibition of adenoviruses.
Asunto(s)
Adenovirus Humanos , Proteínas Argonautas , MicroARNs , Replicación Viral , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/fisiología , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Interferencia de ARN , Células HEK293RESUMEN
The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.
Asunto(s)
Proteínas Argonautas , Conformación de Ácido Nucleico , ARN Guía de Sistemas CRISPR-Cas , Complejo Silenciador Inducido por ARN , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/química , Humanos , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/química , Cinética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Interferencia de ARN , Secuencia de Bases , Células HEK293RESUMEN
Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca2+ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca2+ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E2) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E2 treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E2 treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. NEW AND NOTEWORTHY: In this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.
Asunto(s)
Proteínas Argonautas , MicroARNs , Miocitos Cardíacos , Especies Reactivas de Oxígeno , MicroARNs/genética , MicroARNs/metabolismo , Animales , Femenino , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Masculino , Fosforilación , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Especies Reactivas de Oxígeno/metabolismo , Ratones , Mitocondrias Cardíacas/metabolismo , Calcio/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , Complejo Silenciador Inducido por ARN/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Caracteres SexualesRESUMEN
Eukaryotic Argonaut proteins (AGOs) assemble RNA-induced silencing complexes (RISCs) with guide RNAs that allow binding to complementary RNA sequences and subsequent silencing of target genes. The model plant Arabidopsis thaliana encodes 10 different AGOs, categorized into three distinct clades based on amino acid sequence similarity. While clade 1 and 2 RISCs are known for their roles in post-transcriptional gene silencing, and clade 3 RISCs are associated with transcriptional gene silencing in the nucleus, the specific mechanisms of how RISCs from each clade recognize their targets remain unclear. In this study, I conducted quantitative binding analyses between RISCs and target nucleic acids with mismatches at various positions, unveiling distinct target binding characteristics unique to each clade. Clade 1 and 2 RISCs require base pairing not only in the seed region but also in the 3' supplementary region for stable target RNA binding, with clade 1 exhibiting a higher stringency. Conversely, clade 3 RISCs tolerate dinucleotide mismatches beyond the seed region. Strikingly, they bind to DNA targets with an affinity equal to or surpassing that of RNA, like prokaryotic AGO complexes. These insights challenge existing views on plant RNA silencing and open avenues for exploring new functions of eukaryotic AGOs.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Complejo Silenciador Inducido por ARN , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , ARN de Planta/metabolismo , ARN de Planta/genética , ARN de Planta/química , Unión Proteica , Interferencia de ARN , Disparidad de Par Base , ADN de Plantas/metabolismo , ADN de Plantas/genéticaRESUMEN
In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.
Asunto(s)
ARN Interferente Pequeño , Complejo Silenciador Inducido por ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/química , Humanos , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/química , Fosforilación , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/químicaRESUMEN
Environmental stresses diversely affect multiple processes related to the growth, development, and yield of many crops worldwide. In response, plants have developed numerous sophisticated defense mechanisms at the cellular and subcellular levels to react and adapt to biotic and abiotic stressors. RNA silencing, which is an innate immune mechanism, mediates sequence-specific gene expression regulation in higher eukaryotes. ARGONAUTE (AGO) proteins are essential components of the RNA-induced silencing complex (RISC). They bind to small noncoding RNAs (sRNAs) and target complementary RNAs, causing translational repression or triggering endonucleolytic cleavage pathways. In this review, we aim to illustrate the recently published molecular functions, regulatory mechanisms, and biological roles of AGO family proteins in model plants and cash crops, especially in the defense against diverse biotic and abiotic stresses, which could be helpful in crop improvement and stress tolerance in various plants.
Asunto(s)
Proteínas Argonautas , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas Argonautas/genética , Regulación de la Expresión Génica de las Plantas , Complejo Silenciador Inducido por ARN/genética , Productos Agrícolas/genéticaRESUMEN
MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins in the RNA-induced silencing complex (RISC) to modulate protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by repressing the translation of proteins involved in dendritic spine morphogenesis. Rapid NMDAR-dependent silencing of Limk1 is essential for spine shrinkage and requires Ago2 phosphorylation at S387. Not all gene silencing events are modulated by S387 phosphorylation, and the mechanisms that govern the selection of specific mRNAs for silencing downstream of S387 phosphorylation are unknown. Here, we show that NMDAR-dependent S387 phosphorylation causes a rapid and transient increase in the association of Ago2 with Limk1, but not Apt1 mRNA. The specific increase in Limk1 mRNA binding to Ago2 requires recruitment of the helicase DDX6 to RISC. Furthermore, we show that DDX6 is required for NMDAR-dependent silencing of Limk1 via miR-134, but not Apt1 via miR-138, and is essential for NMDAR-dependent spine shrinkage. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression of specific genes to control dendritic spine morphology.
Asunto(s)
MicroARNs , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Espinas Dendríticas/metabolismo , ARN Helicasas/metabolismo , MicroARNs/genética , Proteínas Argonautas/genética , Complejo Silenciador Inducido por ARN/metabolismo , Silenciador del Gen , ARN Mensajero/genéticaRESUMEN
RNA interference (RNAi) is becoming medicine for curing human diseases. Still, we lack a thorough understanding of some fundamental aspects of RNAi that affect its efficiency and accuracy. One such question is how RNA-induced silencing complex (RISC) can efficiently find its targets. To address this question, we developed a strategy that involves the expression of mRNAs containing concatenations of identical miRNA/siRNA target sites. These mRNAs were cleaved by co-expressed miRNAs in plant cells or by co-transfected siRNAs in mammalian cells. The mRNA cleavage events were then detected using the 5'RACE assay. Using this strategy, we found that RISCs preferentially cleave the upstream ones of concatenated target sites, consistent with a model that RISC scans mRNA in 5'â3' direction to approach its target sites. The stability of the cleaved mRNA fragments correlates with the complementarity between siRNA and its target sequence. When siRNA perfectly complements its target sequence, the cleaved mRNA fragment becomes stable and may be cleaved in a second round. Our findings have practical implications for designing siRNAs with increased efficiency and reduced off-target effects.
Asunto(s)
MicroARNs , Animales , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mamíferos/metabolismoRESUMEN
During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Animales , Humanos , Mamíferos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Viral/genética , ARN Viral/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , ARN Subgenómico , Proteínas Virales/metabolismo , Replicación Viral , Virus Zika/fisiología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Alzheimer's disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA-induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to Aß42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of "SuperAgers", humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to Aß42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and Aß42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration.
Asunto(s)
Enfermedad de Alzheimer , MicroARNs , Ratones , Animales , Humanos , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , MicroARNs/genética , Complejo Silenciador Inducido por ARN/genética , Interferencia de ARN , Envejecimiento/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidadRESUMEN
BACKGROUND: Zika virus congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta without causing overt cellular or tissue damage is fundamental to deciphering mechanisms of maternal-fetal vertical transmission. OBJECTIVE: Placenta-specific microRNAs are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that the Zika virus functionally disrupts placental microRNAs, enabling viral persistence and fetal pathogenesis. STUDY DESIGN: To test this hypothesis, we used orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation to identify any significant alterations in the functional loading of microRNAs and their targets onto the RNA-induced silencing complex. Trophoblasts from same-donors were split and infected with a contemporary first-passage Zika virus strain HN16 (multiplicity of infection=1 plaque forming unit per cell) or mock infected. To functionally cross-validate microRNA-messenger RNA interactions, we compared our Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation results with an independent analysis of published bulk RNA-sequencing data from human placental disk specimens (n=3 subjects; Zika virus positive in first, second, or third trimester, CD45- cells sorted by flow cytometry) and compared it with uninfected controls (n=2 subjects). To investigate the importance of these microRNA and RNA interference networks in Zika virus pathogenesis, we used a gnotobiotic mouse model uniquely susceptible to the Zika virus. We evaluated if small-molecule enhancement of microRNA and RNA interference pathways with enoxacin influenced Zika virus pathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9743 spatial transcriptomes) to identify potential Zika virus-associated alterations in immune microenvironments. RESULTS: We found that Zika virus infection of primary human trophoblast cells led to an unexpected disruption of placental microRNA regulation networks. When compared with uninfected controls, Zika virus-infected placentae had significantly altered SLC12A8, SDK1, and VLDLR RNA-induced silencing complex loading and transcript levels (-2
Asunto(s)
MicroARNs , Infección por el Virus Zika , Virus Zika , Embarazo , Humanos , Femenino , Animales , Ratones , Virus Zika/genética , Infección por el Virus Zika/genética , MicroARNs/genética , MicroARNs/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Enoxacino/metabolismo , Placenta/metabolismo , Perfilación de la Expresión Génica , Complejo Silenciador Inducido por ARN/metabolismo , Factores de Crecimiento Transformadores/metabolismo , Trofoblastos/metabolismoRESUMEN
Triantennary N-acetyl-D galactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) have majorly advanced the development of RNA-based therapeutics. Chemically stabilized GalNAc-siRNAs exhibit extensive albeit capacity-limited (nonlinear) distribution into hepatocytes with additional complexities in intracellular liver disposition and pharmacology. A mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model of GalNAc-siRNA was developed to i) quantitate ASGPR-mediated disposition and downstream RNA-induced silencing complex (RISC)-dependent pharmacology following intravenous (IV) and subcutaneous (SC) dosing, ii) assess the kinetics of formed active metabolite, iii) leverage, as an example, published experimental data for givosiran, and iv) demonstrate PK translation across two preclinical species (rat and monkey) with subsequent prediction of human plasma PK. The structural model is based on competition between parent and formed active metabolite for occupancy and uptake via ASGPR into hepatocytes, intracellular sequestration and degradation, and downstream engagement of RNA-induced silencing complex (RISC) governing target mRNA degradation. The model jointly and accurately captured available concentration-time profiles of givosiran and/or AS(N-1)3' givosiran in rat and/or monkey plasma, liver, and/or kidney following givosiran administered both IV and SC. RISC-dependent gene silencing of ALAS1 mRNA was well-characterized. The model estimated an in vivo affinity (KD) value of 27.7 nM for GalNAc-ASGPR and weight-based allometric exponents of -0.27 and -0.24 for SC absorption and intracellular (endolysosomal) degradation rate constants. The model well-predicted reported givosiran plasma PK profiles in humans. PK simulations revealed net-shifts in liver-to-kidney distribution ratios with increasing IV and SC dose. Importantly, decreases in the relative liver uptake efficiency were demonstrated following IV and, to a lesser extent, following SC dosing explained by differential ASGPR occupancy profiles over time.
Asunto(s)
Galactosamina , Complejo Silenciador Inducido por ARN , Humanos , Ratas , Animales , ARN Interferente Pequeño/genética , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Silenciador del Gen , Haplorrinos/genética , Haplorrinos/metabolismoRESUMEN
Argonaute (AGO) proteins in all three domains of life form ribonucleoprotein or deoxyribonucleoprotein complexes by loading a guide RNA or DNA, respectively. Since all AGOs retain a PIWI domain that takes an RNase H fold, the ancestor was likely an endoribonuclease (i.e., a slicer). In animals, most miRNA-mediated gene silencing occurs slicer independently. However, the slicer activity of AGO is indispensable in specific events, such as development and differentiation, which are critical for vertebrates and thus cannot be replaced by the slicer-independent regulation. This review highlights the distinctions in catalytic activation mechanisms among slicing-competent AGOs, shedding light on the roles of two metal ions in target recognition and cleavage. The precision of the target specificity by the RNA-induced silencing complexes is reevaluated and redefined. The possible coevolutionary relationship between slicer-independent gene regulation and AGO-binding protein, GW182, is also explored. These discussions reveal that numerous captivating questions remain unanswered regarding the timing and manner in which AGOs employ their slicing activity.
Asunto(s)
Proteínas Argonautas , Complejo Silenciador Inducido por ARN , Ribonucleasas , Animales , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Ribonucleasas/química , Ribonucleasas/metabolismo , ARN Guía de Sistemas CRISPR-Cas , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/química , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs). The assembly process establishes the seed, central, 3' supplementary, and tail regions across the loaded guide, enabling the RISC to recognize target RNAs for silencing. This guide segmentation is caused by anchoring the 3' end at the AGO PAZ domain, but the minimum guide length required for the conformation remains to be studied because the current miRNA size defined by Dicer processing is ambiguous. Using a 3' â 5' exonuclease ISG20, we determined the lengths of AGO-associated miR-20a and let-7a with 3' ends that no longer reach the PAZ domain. Unexpectedly, miR-20a and let-7a needed different lengths, 19 and 20 nt, respectively, to maintain their RISC conformation. This difference can be explained by the low affinity of the PAZ domain for the adenosine at g19 of let-7a, suggesting that the tail-region sequence slightly alters the minimum guide length. We also present that 17-nt guides are sufficiently short enough to function as tinyRNAs (tyRNAs) whose 3' ends are not anchored at the PAZ domain. Since tyRNAs do not have the prerequisite anchoring for the standardized guide segmentation, they would recognize targets differently from miRNAs and siRNAs.
Asunto(s)
MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , ARN Bicatenario , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMEN
ARGONAUTE (AGO) proteins are key components of the RNA-induced silencing complex (RISC) that mediates gene silencing in eukaryotes. Small-RNA (sRNA) cargoes are selectively loaded into different members of the AGO protein family and then target complementary sequences to in-duce transcriptional repression, mRNA cleavage, or translation inhibition. Previous reviews have mainly focused on the traditional roles of AGOs in specific biological processes or on the molecular mechanisms of sRNA sorting. In this review, we summarize the biological significance of canonical sRNA loading, including the balance among distinct sRNA pathways, cross-regulation of different RISC activities during plant development and defense, and, especially, the emerging roles of AGOs in sRNA movement. We also discuss recent advances in novel non-canonical functions of plant AGOs. Perspectives for future functional studies of this evolutionarily conserved eukaryotic protein family will facilitate a more comprehensive understanding of the multi-faceted AGO proteins.
Asunto(s)
MicroARNs , ARN Pequeño no Traducido , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Complejo Silenciador Inducido por ARN/genética , ARN Pequeño no Traducido/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Increasing evidence suggests mammalian Argonaute (Ago) proteins partition into distinct complexes within cells, but there is still little biochemical or functional understanding of the miRNAs differentially associated with these complexes. In naïve T cells, Ago2 is found almost exclusively in low molecular weight (LMW) complexes which are associated with miRNAs but not their target mRNAs. Upon T-cell activation, a proportion of these Ago2 complexes move into a newly formed high molecular weight (HMW) RNA-induced silencing complex (RISC), which is characterized by the presence of the GW182 protein that mediates translational repression. Here, we demonstrate distinct partitioning of miRNAs and isomiRs in LMW versus HMW RISCs upon antigen-mediated activation of CD8+ T cells. We identify miR-7 as highly enriched in HMW RISC and demonstrate that miR-7 inhibition leads to increased production of IL-2 and up-regulation of the IL-2 receptor, the transferrin receptor, CD71 and the amino acid transporter, CD98. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signaling and the metabolic processes regulated by IL-2.
Asunto(s)
MicroARNs , Complejo Silenciador Inducido por ARN , Animales , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Linfocitos T CD8-positivos/metabolismo , Peso Molecular , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Mamíferos/metabolismoRESUMEN
MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing.