RESUMEN
DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.
Asunto(s)
ARN Helicasas DEAD-box , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/química , Humanos , Animales , ARN/metabolismo , ARN/genética , ARN/química , Empalme del ARN , Orgánulos/metabolismo , Orgánulos/genética , Ribosomas/metabolismo , Ribosomas/genética , Pliegue del ARN , Procesamiento Postranscripcional del ARN , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.
Asunto(s)
Envejecimiento , Encéfalo , Complemento C1q , Homeostasis , Microglía , Neuronas , Ribonucleoproteínas , Animales , Complemento C1q/metabolismo , Ratones , Microglía/metabolismo , Envejecimiento/metabolismo , Encéfalo/metabolismo , Ribonucleoproteínas/metabolismo , Neuronas/metabolismo , Ratones Endogámicos C57BL , HumanosRESUMEN
Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.
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Autoanticuerpos , Enfermedades Autoinmunes , ARN Largo no Codificante , Animales , Femenino , Humanos , Masculino , Ratones , Autoanticuerpos/genética , Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismo , Inactivación del Cromosoma X , Caracteres SexualesRESUMEN
mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
Asunto(s)
ARN Mensajero , Proteínas de Unión al ARN , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Unión Proteica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Células HeLa , Factores de Tiempo , Aprendizaje AutomáticoRESUMEN
The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
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Citoplasma , Polirribosomas , Ribonucleoproteínas , Polirribosomas/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulación de Dinámica Molecular , ARN Mensajero/metabolismo , ARN Mensajero/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Condensados Biomoleculares/metabolismo , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genéticaRESUMEN
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
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Factor 4F Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Respuesta al Choque Térmico , Proteínas de Unión a Poli(A) , Biosíntesis de Proteínas , ARN Mensajero , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Respuesta al Choque Térmico/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Regulación Fúngica de la Expresión Génica , Unión Proteica , ARN de Hongos/metabolismo , ARN de Hongos/genéticaRESUMEN
TDP-43 is an essential RNA-binding protein strongly implicated in the pathogenesis of neurodegenerative disorders characterized by cytoplasmic aggregates and loss of nuclear TDP-43. The protein shuttles between nucleus and cytoplasm, yet maintaining predominantly nuclear TDP-43 localization is important for TDP-43 function and for inhibiting cytoplasmic aggregation. We previously demonstrated that specific RNA binding mediates TDP-43 self-assembly and biomolecular condensation, requiring multivalent interactions via N- and C-terminal domains. Here, we show that these complexes play a key role in TDP-43 nuclear retention. TDP-43 forms macromolecular complexes with a wide range of size distribution in cells and we find that defects in RNA binding or inter-domain interactions, including phase separation, impair the assembly of the largest species. Our findings suggest that recruitment into these macromolecular complexes prevents cytoplasmic egress of TDP-43 in a size-dependent manner. Our observations uncover fundamental mechanisms controlling TDP-43 cellular homeostasis, whereby regulation of RNA-mediated self-assembly modulates TDP-43 nucleocytoplasmic distribution. Moreover, these findings highlight pathways that may be implicated in TDP-43 proteinopathies and identify potential therapeutic targets.
Asunto(s)
Proteínas de Unión al ADN , Ribonucleoproteínas , Proteinopatías TDP-43 , Humanos , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/metabolismo , Sustancias Macromoleculares/metabolismo , Ribonucleoproteínas/metabolismo , ARN , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/metabolismoRESUMEN
Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.
Asunto(s)
Comercio , Edición Génica , Terapia Genética , ARN Mensajero , RibonucleoproteínasRESUMEN
Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.
Asunto(s)
Ataxina-2 , Proteínas de Drosophila , Drosophila melanogaster , ARN Mensajero , Ribonucleoproteínas , Ataxina-2/genética , Ataxina-2/metabolismo , Animales , Humanos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/genética , Animales Modificados Genéticamente , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ADNRESUMEN
Cells contain a myriad of membraneless ribonucleoprotein (RNP) condensates with distinct compositions of proteins and RNAs. RNP condensates participate in different cellular activities, including RNA storage, mRNA translation or decay, stress response, etc. RNP condensates are assembled via liquid-liquid phase separation (LLPS) driven by multivalent interactions. Transition of RNP condensates into bodies with abnormal material properties, such as solid-like amyloid structures, is associated with the pathogenesis of various diseases. In this review, we focus on how RNAs regulate multiple aspects of RNP condensates, such as dynamic assembly and/or disassembly and biophysical properties. RNA properties - including concentration, sequence, length and structure - also determine the phase behaviors of RNP condensates. RNA is also involved in specifying autophagic degradation of RNP condensates. Unraveling the role of RNA in RNPs provides novel insights into pathological accumulation of RNPs in various diseases. This new understanding can potentially be harnessed to develop therapeutic strategies.
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Condensados Biomoleculares , ARN , ARN/genética , Ribonucleoproteínas/metabolismo , AutofagiaRESUMEN
L1TD1 is a cytoplasmic RNA-binding protein specifically expressed in pluripotent stem cells and, unlike its mouse ortholog, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known protein-coding gene domesticated from a LINE-1 (L1) retroelement, the functional legacy of its ancestral protein, ORF1p of L1, and how it is manifested in L1TD1 are still unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs and localizes to high-density ribonucleoprotein (RNP) condensates. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. L1TD1 depletion promoted the formation of stress granules in embryonic stem cells. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain of L1TD1 facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatment of neurodegenerative diseases caused by disturbed stress granule dynamics.
Asunto(s)
Células Madre Embrionarias , Proteínas de Unión al ARN , Ribonucleoproteínas , Animales , Humanos , Ratones , Gránulos Citoplasmáticos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células HeLa , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , Multimerización de Proteína , ARN Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , Unión Proteica , Sitios de Unión , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ensamble de Virus/genética , Humanos , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , COVID-19/virologíaRESUMEN
Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.
Asunto(s)
Cilios , Ribonucleoproteínas , Tubulina (Proteína) , Proteínas de Xenopus , Xenopus laevis , Cilios/metabolismo , Animales , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Humanos , Antígeno SS-B , Autoantígenos/metabolismo , Autoantígenos/genética , Unión Proteica , Axonema/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.
Asunto(s)
Anexina A3 , Hepacivirus , Hepatitis C , Antígeno SS-B , Internalización del Virus , Humanos , Anexina A3/metabolismo , Anexina A3/genética , Autoantígenos/metabolismo , Autoantígenos/genética , Células HEK293 , Hepacivirus/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Hepatitis C/genética , Interacciones Huésped-Patógeno , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Genotipo , Lípidos , Protoplastos , Vitis , Vitis/genética , Edición Génica/métodos , Protoplastos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Vino , Genoma de Planta/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ribonucleoproteínas , Edición Génica/métodos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Leishmania/genética , Leishmania/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismo , TransfecciónRESUMEN
The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.
Asunto(s)
Virus de la Hepatitis B , Factor Nuclear 4 del Hepatocito , Hepatocitos , Interferón gamma , Ribonucleoproteínas , Replicación Viral , Humanos , Virus de la Hepatitis B/fisiología , Animales , Ratones , Interferón gamma/farmacología , Interferón gamma/metabolismo , Hepatocitos/virología , Hepatocitos/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hepatitis B/virología , Hepatitis B/metabolismo , Células Hep G2 , Línea Celular TumoralRESUMEN
The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Nanopartículas , Polímeros , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Humanos , Polímeros/química , Nanopartículas/química , Animales , Ribonucleoproteínas/genética , Ribonucleoproteínas/química , Células HEK293 , Ratones , Guanidinas/químicaRESUMEN
Messenger ribonucleoprotein particles (mRNPs) are vital for tissue-specific gene expression via mediating posttranscriptional regulations. However, proteomic profiling of proteins in mRNPs, i.e., mRNA-associated proteins (mRAPs), has been challenging at the tissue level. Herein, we report the development of formaldehyde cross-linking-based mRNA-associated protein profiling (FAXRAP), a chemical strategy that enables the identification of mRAPs in both cultured cells and intact mouse organs. Applying FAXRAP, tissue-specific mRAPs were systematically profiled in the mouse liver, kidney, heart, and brain. Furthermore, brain mRAPs in Parkinson's disease (PD) mouse model were investigated, which revealed a global decrease of mRNP assembly in the brain of mice with PD. We envision that FAXRAP will facilitate uncovering the posttranscriptional regulation networks in various biological systems.
Asunto(s)
Proteómica , Ribonucleoproteínas , Ratones , Animales , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , FormaldehídoRESUMEN
Atherosclerosis, characterized by the accumulation of lipid plaques on the inner walls of arteries, is the leading cause of heart attack, stroke and severe ischemic injuries. Senescent cells have been found to accumulate within atherosclerotic lesions and contribute to the progression of atherosclerosis. In our previous study, we discovered that suppressing Larp7 accelerates senescence by inhibiting Sirt1 activity, resulting in increased atherosclerosis in high-fat diet (HFD) fed and ApoE deficient (ApoEKO) mice. However, there has been no direct evidence demonstrating Larp7 per se could attenuate atherosclerosis. To this end, we generated a tetO-controlled and Cre-activated Larp7 gain-of-function mouse. Through RT-PCR and western blotting, we confirmed Larp7 overexpression in the aortas of HFD-fed ApoEKO; Larp7tetO mice. Larp7 overexpression led to increased Sirt1 activity and decreased cellular senescence signals mediated by p53/p65 in the aortas. Additionally, Larp7 overexpression reduced the presence of p16-positive senescent cells in the aortic lesions. Furthermore, Larp7 overexpression resulted in a decrease in pro-inflammatory macrophages and SASP factors. Consequently, Larp7 overexpression led to a reduction in the area of atherosclerotic lesions in HFD-fed ApoEKO; Larp7tetO mice. In summary, our study provides evidence that Larp7 overexpression holds promise as an approach to inhibit cellular senescence and prevent atherosclerosis.