RESUMO
Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.
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Retrovirus Endógenos/genética , Histona Desmetilases/metabolismo , Complexo de Inativação Induzido por RNA/genética , Animais , Linhagem Celular Tumoral , Cromatina , Terapia Combinada , Regulação da Expressão Gênica/genética , Histona Desmetilases/genética , Humanos , Imunidade Celular , Imunoterapia , Interferon Tipo I , Células MCF-7 , Camundongos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , RNA de Cadeia Dupla/genética , Linfócitos TRESUMO
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.
Assuntos
Proteínas Argonautas , Conformação de Ácido Nucleico , RNA Guia de Sistemas CRISPR-Cas , Complexo de Inativação Induzido por RNA , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/química , Humanos , Complexo de Inativação Induzido por RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/química , Cinética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Interferência de RNA , Sequência de Bases , Células HEK293RESUMO
Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes). These mechanisms can allow specific destruction of a regulatory RNA via pairing with a decay trigger RNA or function as global off switches by disrupting the stability or function of the protein partner.
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Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.
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Proteínas Argonautas/química , Modelos Químicos , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/química , Animais , CamundongosRESUMO
In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.
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Proteínas Argonautas/fisiologia , Inativação Gênica/fisiologia , MicroRNAs/fisiologia , Animais , Proteínas Argonautas/genética , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular , Citoplasma , Células-Tronco Embrionárias/metabolismo , Humanos , Mamíferos , Camundongos , MicroRNAs/genética , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de TranscriçãoRESUMO
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.
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Proteínas Argonautas/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 Argonautas/genética , Proteínas Argonautas/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 RNARESUMO
Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3'UTR with two regions of AID-dependent Ago2-binding sites (5'- and 3'dABs). Top1 3'UTR knockout (3'UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3'UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3'UTR-mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5'- and 3'dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3'UTRKO cells to wild-type cells whereas it does not impact on 3'UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3'UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.
Assuntos
MicroRNAs , MicroRNAs/genética , Regiões 3' não Traduzidas , Clivagem do DNA , Citidina Desaminase/genética , Switching de Imunoglobulina , Anticorpos/genética , Hipermutação Somática de ImunoglobulinaRESUMO
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.
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Proteínas Argonautas , Complexo de Inativação Induzido por RNA , Ribonucleases , Animais , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , RNA Guia de Sistemas CRISPR-Cas , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
Assuntos
MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Interferência de RNA , Proteínas Argonautas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
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MicroRNAs , Complexo de Inativação Induzido por RNA , Animais , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Peso Molecular , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Mamíferos/metabolismoRESUMO
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.
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Infecção por Zika virus , Zika virus , Animais , Humanos , Mamíferos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/genética , RNA Viral/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , RNA Subgenômico , Proteínas Virais/metabolismo , Replicação Viral , Zika virus/fisiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
The RNAi pathway provides both innate immunity and efficient gene-knockdown tools in many eukaryotic species, but curiously not in zebrafish. We discovered that RNAi is less effective in zebrafish at least partly because Argonaute2-catalyzed mRNA slicing is impaired. This defect is due to two mutations that arose in an ancestor of most teleost fish, implying that most fish lack effective RNAi. Despite lacking efficient slicing activity, these fish have retained the ability to produce miR-451, a microRNA generated by a cleavage reaction analogous to slicing. This ability is due to a G-G mismatch within the fish miR-451 precursor, which substantially enhances its cleavage. An analogous G-G mismatch (or sometimes also a G-A mismatch) enhances target slicing, despite disrupting seed pairing important for target binding. These results provide a strategy for restoring RNAi to zebrafish and reveal unanticipated opposing effects of a seed mismatch with implications for mechanism and guide-RNA design.
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Proteínas Argonautas/metabolismo , Pareamento Incorreto de Bases , MicroRNAs/metabolismo , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , Peixe-Zebra/genética , Animais , Proteínas Argonautas/genética , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Peixe-Zebra/fisiologiaRESUMO
While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of â¼50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins.
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Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , MicroRNAs/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , Transativadores/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , Análise de Sequência de RNA , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
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.
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MicroRNAs , Animais , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mamíferos/metabolismoRESUMO
It is of great importance and worthy of efforts to give a clear structure-property relationship and microscopic mechanism of fluorescence emitters with high quantum yield. In this work, we perform a detailed computational investigation to give an explanation to the high efficiency of a fluorescence emitter XBTD-NPh based TADF sensitized fluorescence (TSF) OLEDs, and construct a symmetry structure DSBNA-BTD. Theoretical calculations show that XBTD-NPh is a long-time phosphorescent material at 77 K and TADF is attributed to the RISC of T1 to S1 state. For DSBNA-BTD, excitons arrived at T1 state comes to a large rate of nonradiatively path to the ground state, meaning it is may not be an efficient TADF molecule. For both molecules, the fast IC between T2 and T1 state results in that the hot exciton channel T1-Tn-S1 makes no contribution to the TADF.
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HIV-1 (HIV) infects CD4+ T cells, the gradual depletion of which can lead to AIDS in the absence of antiretroviral therapy (ART). Some cells, however, survive HIV infection and persist as part of the latently infected reservoir that causes recurrent viremia after ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could lead to a way to clear the latent reservoir. Death induced by survival gene elimination (DISE), an RNA interference (RNAi)-based mechanism, kills cells through short RNAs (sRNAs) with toxic 6-mer seeds (positions 2 to 7 of sRNA). These toxic seeds target the 3' untranslated region (UTR) of mRNAs, decreasing the expression of hundreds of genes critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded nontoxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. HIV has been shown to inhibit the biogenesis of host miRNAs in multiple ways. We now report that HIV infection of cells deficient in miRNA expression or function results in enhanced RISC loading of an HIV-encoded miRNA HIV-miR-TAR-3p, which can kill cells by DISE through a noncanonical (positions 3 to 8) 6-mer seed. In addition, cellular RISC-bound sRNAs shift to lower seed viability. This also occurs after latent HIV provirus reactivation in J-Lat cells, suggesting independence of permissiveness of cells to viral infection. More precise targeting of the balance between protective and cytotoxic sRNAs could provide new avenues to explore novel cell death mechanisms that could be used to kill latent HIV. IMPORTANCE Several mechanisms by which initial HIV infection is cytotoxic to infected cells have been reported and involve various forms of cell death. Characterizing the mechanisms underlying the long-term survival of certain T cells that become persistent provirus reservoirs is critical to developing a cure. We recently discovered death induced by survival gene elimination (DISE), an RNAi-based mechanism of cell death whereby toxic short RNAs (sRNAs) containing 6-mer seed sequences (exerting 6-mer seed toxicity) targeting essential survival genes are loaded into RNA-induced silencing complex (RISC) complexes, resulting in inescapable cell death. We now report that HIV infection in cells with low miRNA expression causes a shift of mostly cellular RISC-bound sRNAs to more toxic seeds. This could prime cells to DISE and is further enhanced by the viral microRNA (miRNA) HIV-miR-TAR-3p, which carries a toxic noncanonical 6-mer seed. Our data provide multiple new avenues to explore novel cell death mechanisms that could be used to kill latent HIV.
Assuntos
Infecções por HIV , HIV-1 , MicroRNAs , Humanos , HIV-1/fisiologia , Latência Viral/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
The establishment of the Orsay virus-Caenorhabditis elegans infection model has enabled the identification of host factors essential for virus infection. Argonautes are RNA interacting proteins evolutionary conserved in the three domains of life that are key components of small RNA pathways. C. elegans encodes 27 argonautes or argonaute-like proteins. Here, we determined that mutation of the argonaute-like gene 1, alg-1, results in a greater than 10,000-fold reduction in Orsay viral RNA levels, which could be rescued by ectopic expression of alg-1. Mutation in ain-1, a known interactor of ALG-1 and component of the RNA-induced silencing complex, also resulted in a significant reduction in Orsay virus levels. Viral RNA replication from an endogenous transgene replicon system was impaired by the lack of ALG-1, suggesting that ALG-1 plays a role during the replication stage of the virus life cycle. Orsay virus RNA levels were unaffected by mutations in the ALG-1 RNase H-like motif that ablate the slicer activity of ALG-1. These findings demonstrate a novel function of ALG-1 in promoting Orsay virus replication in C. elegans. IMPORTANCE All viruses are obligate intracellular parasites that recruit the cellular machinery of the host they infect to support their own proliferation. We used Caenorhabditis elegans and its only known infecting virus, Orsay virus, to identify host proteins relevant for virus infection. We determined that ALG-1, a protein previously known to be important in influencing worm life span and the expression levels of thousands of genes, is required for Orsay virus infection of C. elegans. This is a new function attributed to ALG-1 that was not recognized before. In humans, it has been shown that AGO2, a close relative protein to ALG-1, is essential for hepatitis C virus replication. This demonstrates that through evolution from worms to humans, some proteins have maintained similar functions, and consequently, this suggests that studying virus infection in a simple worm model has the potential to provide novel insights into strategies used by viruses to proliferate.
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Proteínas de Caenorhabditis elegans , Nodaviridae , Animais , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/virologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Nodaviridae/genética , Nodaviridae/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/genética , Infecções por Vírus de RNA/virologia , MutaçãoRESUMO
Electrochemiluminescence (ECL) featuring thermally activated delayed fluorescence (TADF) properties has attracted considerable interest, showcasing their potential for 100 % exciton harvesting, which marks a significant advancement in the realm of organic ECL. However, the challenge of elucidating the precise contribution of TADF to the enhanced ECL efficiency arises due to the lack of comparative studies of organic compounds with or without efficient TADF properties. In this study, we present four carbazole-benzonitrile molecules possessing similar chemical structures and comparable exchange energy (ΔEST). Despite their comparable properties, these compounds exhibited varying TADF efficiencies, warranting a closer examination of their underlying structural and electronic characteristics governing the optical properties. Consequently, intense ECL emission was only observed from 4CzBN with a remarkable TADF efficiency, underscoring the substantial difference in the ECL signal among molecules with comparable ΔEST and similar spectral properties but varying TADF activity.
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This study conducts a thorough theoretical investigation of Thermally Activated Delayed Fluorescence (TADF) in phenothiazine-based systems, examining ten molecular configurations recognized experimentally as TADF-active. Employing Time-Dependent Density Functional Theory (TD-DFT), our analysis spans the investigation of singlet-triplet energy gaps (ΔEST), spin-orbit coupling, and excitation characteristics using Multiwfn. This approach not only validates the adherence to El Sayed's rule across these systems but also provides a detailed understanding of charge transfer dynamics, as visualized through heat maps. A significant aspect of our study is the exploration of different oxidation states of sulfur and site substitutions on phenothiazine. This systematic variation aims to identify additional TADF-active compounds, drawing parallels with properties characterizing other known TADF emitters. Our investigation into Reverse Intersystem Crossing (rISC) rates and the analysis of dihedral angles in relation to ΔEST values offer nuanced insights into the TADF behaviours of these molecules. By integrating rigorous computational analysis with practical implications, we provide a foundational understanding that enhances the design and optimization of phenothiazine-based materials for optoelectronic applications. This work not only advances our theoretical understanding of TADF in phenothiazine derivatives but also serves as a guide for experimentalists and industry professionals in the strategic design of new TADF materials.
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During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFß) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.