RESUMO
To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. including some of the researchers involved in the 2013 work investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.
Assuntos
RNA Polimerase II , RNA Mensageiro , Fatores de Transcrição , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estabilidade de RNA , Regiões Promotoras Genéticas , Ligação Proteica , Dedos de Zinco , Transcrição Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Citoplasma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
The main forces driving protein complex evolution are currently not well understood, especially in homomers, where quaternary structure might frequently evolve neutrally. Here we examine the factors determining oligomerisation by analysing the evolution of enzymes in circumstances where homomers rarely evolve. We show that 1) In extracellular environments, most enzymes with known structure are monomers, while in the cytoplasm homomers, indicating that the evolution of oligomers is cellular environment dependent; 2) The evolution of quaternary structure within protein orthogroups is more consistent with the predictions of constructive neutral evolution than an adaptive process: quaternary structure is gained easier than it is lost, and most extracellular monomers evolved from proteins that were monomers also in their ancestral state, without the loss of interfaces. Our results indicate that oligomerisation is context-dependent, and even when adaptive, in many cases it is probably not driven by the intrinsic properties of enzymes, like their biochemical function, but rather the properties of the environment where the enzyme is active. These factors might be macromolecular crowding and excluded volume effects facilitating the evolution of interfaces, and the maintenance of cellular homeostasis through shaping cytoplasm fluidity, protein degradation, or diffusion rates.
Assuntos
Citoplasma , Enzimas , Evolução Molecular , Estrutura Quaternária de Proteína , Enzimas/química , Enzimas/metabolismo , Enzimas/genética , Citoplasma/metabolismo , Multimerização ProteicaRESUMO
N6-methyladenosine (m6A) is the most abundant post-transcriptional dynamic RNA modification process in eukaryotes, extensively implicated in cellular growth, embryonic development and immune homeostasis. One of the most profound biological functions of m6A is to regulate RNA metabolism, thereby determining the fate of RNA. Notably, the regulation of m6A-mediated organized RNA metabolism critically relies on the assembly of membraneless organelles (MLOs) in both the nucleus and cytoplasm, such as nuclear speckles, stress granules and processing bodies. In addition, m6A-associated MLOs exert a pivotal role in governing diverse RNA metabolic processes encompassing transcription, splicing, transport, decay and translation. However, emerging evidence suggests that dysregulated m6A levels contribute to the formation of pathological condensates in a range of human diseases, including tumorigenesis, reproductive diseases, neurological diseases and respiratory diseases. To date, the molecular mechanism by which m6A regulates the aggregation of biomolecular condensates associated with RNA metabolism is unclear. In this review, we comprehensively summarize the updated biochemical processes of m6A-associated MLOs, particularly focusing on their impact on RNA metabolism and their pivotal role in disease development and related biological mechanisms. Furthermore, we propose that m6A-associated MLOs could serve as predictive markers for disease progression and potential drug targets in the future.
Assuntos
Adenosina , RNA , Humanos , Adenosina/metabolismo , Adenosina/análogos & derivados , RNA/metabolismo , Organelas/metabolismo , Animais , Processamento Pós-Transcricional do RNA , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Núcleo Celular/metabolismo , Citoplasma/metabolismoRESUMO
How intracellular bacteria subvert the major histocompatibility complex (MHC) class I pathway is poorly understood. Here, we show that the obligate intracellular bacterium Orientia tsutsugamushi uses its effector protein, Ank5, to inhibit nuclear translocation of the MHC class I gene transactivator, NLRC5, and orchestrate its proteasomal degradation. Ank5 uses a tyrosine in its fourth ankyrin repeat to bind the NLRC5 N-terminus while its F-box directs host SCF complex ubiquitination of NLRC5 in the leucine-rich repeat region that dictates susceptibility to Orientia- and Ank5-mediated degradation. The ability of O. tsutsugamushi strains to degrade NLRC5 correlates with ank5 genomic carriage. Ectopically expressed Ank5 that can bind but not degrade NLRC5 protects the transactivator during Orientia infection. Thus, Ank5 is an immunoevasin that uses its bipartite architecture to rid host cells of NLRC5 and reduce surface MHC class I molecules. This study offers insight into how intracellular pathogens can impair MHC class I expression.
Assuntos
Antígenos de Histocompatibilidade Classe I , Peptídeos e Proteínas de Sinalização Intracelular , Orientia tsutsugamushi , Orientia tsutsugamushi/metabolismo , Orientia tsutsugamushi/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Citoplasma/metabolismo , Células HEK293 , Proteólise , Tifo por Ácaros/imunologia , Tifo por Ácaros/microbiologia , Tifo por Ácaros/metabolismo , Camundongos , Ubiquitinação , Interações Hospedeiro-Patógeno/imunologiaRESUMO
Immune-mediated liver injury is a common characteristic of various liver diseases, including autoimmune and viral hepatitis. Here, we investigated the role of DEAD-box helicase 3, X-linked (DDX3X) in immune-mediated liver injury. Liver injury was induced in C57BL/6J mice via concanavalin A (Con A). DDX3X hepatocyte-specific knockout (DDX3XΔHep) mice and control (DDX3Xfl/fl) mice were utilized to investigate the role of DDX3X in liver injury. Primary hepatocytes were treated with tunicamycin (TM) to induce ER stress in vitro. The expression of DDX3X in patients with various liver diseases was evaluated. Hepatic DDX3X expression increased, and DDX3X translocated from the cytoplasm to the nucleus during Con A-induced liver injury. DDX3X deficiency ameliorated mouse liver injury and reduced ER stress in liver tissue. The inhibition of ER stress with 4-PBA significantly attenuated liver injury while decreasing DDX3X levels in liver tissue. However, the upregulation of hepatic DDX3X expression reversed Con A-induced liver injury and negated the protective effect of 4-PBA. Mechanistically, the nuclear translocation of DDX3X promoted ER stress-induced apoptosis through the transcriptional induction of CHOP. Moreover, DDX3X was elevated and translocated into the nucleus in patients with HBV-LF and AIH. Additionally, serum DDX3X levels markedly increased in patients with HBV-LF, and a consistent decrease in DDX3X was associated with a good prognosis. The cytoplasmic-to-nuclear translocation of DDX3X promotes ER stress-induced apoptosis, which is an obligatory step that drives hepatic necrosis and tissue damage. Notably, DDX3X is a potential therapeutic target for immune-mediated liver injury.
Assuntos
RNA Helicases DEAD-box , Estresse do Retículo Endoplasmático , Hepatócitos , Camundongos Endogâmicos C57BL , Animais , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Camundongos , Hepatócitos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Camundongos Knockout , Fígado/metabolismo , Fígado/patologia , Fígado/lesões , Masculino , Transporte Ativo do Núcleo Celular , Apoptose/efeitos dos fármacos , Concanavalina ARESUMO
Subcellular protein localization regulates protein function and can be corrupted in cancers1 and neurodegenerative diseases2,3. The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43ΔNLS and FUSR495X. TRAM-mediated relocalization of FUSR495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration4. The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring.
Assuntos
Transporte Proteico , Humanos , Animais , Camundongos , Núcleo Celular/metabolismo , Ligantes , Proteínas Cromossômicas não Histona/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Masculino , Neurônios/metabolismoRESUMO
The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response is mediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further show that the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer.
Assuntos
Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Humanos , Camundongos , Aminoácidos Aromáticos/metabolismo , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais , Transporte Proteico , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells. Leveraging structure refinement with cross-linking restraints, we identify subcellular-specific conformation heterogeneity of PTEN, revealing dynamic differences in its dual specificity domains between the nucleus and cytoplasm. Furthermore, by discerning conformational disparities, we identify 83 cytoplasm-exclusive and 109 nucleus-exclusive PTEN-interacting proteins, each associated with distinct biological functions. Upon induction of ubiquitin-proteasome system stress, we observe dynamic alterations in PTEN assembly and its interacting partners during translocation. These changes, including the identification of components and interaction sites, are characterized using the SPACX approach. Notably, SPACX enables identification of unique interacting proteins specific to PTEN isoforms, including PTEN and PTEN-Long, through the determination of sequence-specific cross-linking interfaces. These findings underscore the potential of SPACX to elucidate the functional diversity of proteins within distinct subcellular sociology.
Assuntos
Reagentes de Ligações Cruzadas , PTEN Fosfo-Hidrolase , Conformação Proteica , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/química , Humanos , Reagentes de Ligações Cruzadas/química , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
CARM1 is predominantly localized in the nucleus and plays a pivotal role in maintaining mitochondrial homeostasis by regulating gene expression. It suppresses mitochondrial biogenesis by downregulating PGC-1α and TFAM expression, while promoting mitochondrial fission through increased DNM1L expression. Under oxidative stress, CARM1 translocates to the cytoplasm, where it directly methylates DRP1 and accelerates mitochondrial fission, enhancing reactive oxygen species (ROS) production. Cytoplasmic localization of CARM1 is facilitated by its phosphorylation at S595 by ROS-activated p38γ MAPK, creating a positive feedback loop. Consequently, cytoplasmic CARM1 contributes to cellular senescence by altering mitochondrial dynamics and increasing ROS levels. This observation was supported by the increased cytoplasmic CARM1 levels and disrupted mitochondrial dynamics in the transformed 10T1/2 cells. Moreover, CARM1 inhibitors not only inhibit the proliferation of cancer cells but also induce apoptotic death in senescent cells. These findings highlight the potential of CARM1 inhibitors, particularly those targeting cytoplasmic functions, as novel strategies for eliminating cancer and senescent cells.
Assuntos
Senescência Celular , Proteína-Arginina N-Metiltransferases , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Humanos , Fosforilação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Dinâmica Mitocondrial , Mitocôndrias/metabolismo , Citoplasma/metabolismo , Estresse Oxidativo , Apoptose , Proliferação de Células , CamundongosRESUMO
Cytoplasmic male sterility (CMS) is important for commercial hybrid seed production. However, it is still not used in eggplant (Solanum melongena L.), and corresponding regulatory genes and mechanisms of action have not been reported. We report CMS line 327A, which was derived from the hybridization between cultivated and wild eggplants. By looking at different stages of anther development under a microscope, we saw that the 327A anther's tapetum layer vacuolized during meiosis, which caused abortion. To investigate the 327A CMS regulatory genes, the mitochondrial genomes of 327A and its maintainer line 327B were assembled de novo. It was found that 15 unique ORFs (Open Reading Frame) were identified in 327A. RT-PCR and RT-QPCAR tests confirmed that orf312a and orf172a, 327A-specific ORFs with a transmembrane domain, were strongly expressed in sterile anthers of 327A. In addition, orf312a has a chimeric structure with the ribosomal protein subunit rpl16. Therefore, orf312a and orf172a can be considered strong candidate genes for CMS. Concurrently, we analyzed the characteristics of CMS to develop a functional molecular marker, CMS312, targeting a future theoretical basis for eggplant CMS three-line molecular breeding.
Assuntos
Genoma Mitocondrial , Infertilidade das Plantas , Solanum melongena , Solanum melongena/genética , Infertilidade das Plantas/genética , Fases de Leitura Aberta/genética , Regulação da Expressão Gênica de Plantas , Citoplasma/genética , Citoplasma/metabolismo , Genes de PlantasRESUMO
CircRNAs are an important class of RNAs with diverse cellular functions in human physiology and disease. A thorough knowledge of circRNAs including their biogenesis and subcellular distribution is important to understand their roles in a wide variety of processes. However, the analysis of circRNAs from total RNA sequencing data remains challenging. Therefore, we developed Calcifer, a versatile workflow for circRNA annotation. Using Calcifer, we analysed APEX-Seq data to compare circRNA occurrence between whole cells, nucleus and subnuclear compartments. We generally find that circRNAs show higher abundance in whole cells compared to nuclear samples, consistent with their accumulation in the cytoplasm. The notable exception is the single-exon circRNA circCANX(9), which is unexpectedly enriched in the nucleus. In addition, we observe that circFIRRE prevails over the linear lncRNA FIRRE in both the cytoplasm and the nucleus. Zooming in on the subnuclear compartments, we show that circRNAs are strongly depleted from nuclear speckles, indicating that excess splicing factors in this compartment counteract back-splicing. Our results thereby provide valuable insights into the subnuclear distribution of circRNAs. Regarding circRNA function, we surprisingly find that the majority of all detected circRNAs possess complete open reading frames with potential for cap-independent translation. Overall, we show that Calcifer is an easy-to-use, versatile and sustainable workflow for the annotation of circRNAs which expands the repertoire of circRNA tools and allows to gain new insights into circRNA distribution and function.
Assuntos
Núcleo Celular , RNA Circular , RNA Circular/genética , RNA Circular/metabolismo , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Citoplasma/metabolismo , Citoplasma/genética , Fases de Leitura Aberta , Anotação de Sequência Molecular , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Splicing de RNA , Biologia Computacional/métodos , Análise de Sequência de RNARESUMO
The coordinated action of transcriptional and post-transcriptional machineries shapes gene expression programs at steady state and determines their concerted response to perturbations. We have developed Nanodynamo, an experimental and computational workflow for quantifying the kinetic rates of nuclear and cytoplasmic steps of the RNA life cycle. Nanodynamo is based on mathematical modelling following sequencing of native RNA from cellular fractions and polysomes. We have applied this workflow to triple-negative breast cancer cells, revealing widespread post-transcriptional RNA processing that is mutually exclusive with its co-transcriptional counterpart. We used Nanodynamo to unravel the coupling between transcription, processing, export, decay and translation machineries. We have identified a number of coupling interactions within and between the nucleus and cytoplasm that largely contribute to coordinating how cells respond to perturbations that affect gene expression programs. Nanodynamo will be instrumental in unravelling the determinants and regulatory processes involved in the coordination of gene expression responses.
Assuntos
Núcleo Celular , Humanos , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , RNA/metabolismo , RNA/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Processamento Pós-Transcricional do RNA , Citoplasma/metabolismo , Cinética , Polirribossomos/metabolismo , Transcrição Gênica , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.
Assuntos
Membrana Celular , Peptídeos Penetradores de Células , Citoplasma , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Membrana Celular/metabolismo , Humanos , Citoplasma/metabolismo , Sistemas de Liberação de Medicamentos , Células HeLa , AnimaisRESUMO
Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom-up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.
Assuntos
Células Artificiais , Núcleo Celular , Biologia Sintética , Biologia Sintética/métodos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Células Artificiais/metabolismo , Transdução de Sinais , Citoplasma/metabolismo , Comunicação CelularRESUMO
Grass carp reovirus (GCRV) is the most virulent pathogen in the genus Aquareovirus, belonging to the family Spinareoviridae. Members of the Spinareoviridae family are known to replicate and assemble in cytoplasmic inclusion bodies termed viroplasms; however, the detailed mechanism underlying GCRV viroplasm formation and its specific roles in virus infection remains largely unknown. Here, we demonstrate that GCRV viroplasms form through liquid-liquid phase separation (LLPS) of the nonstructural protein NS80 and elucidate the specific role of LLPS during reovirus infection and immune evasion. We observe that viroplasms coalesce within the cytoplasm of GCRV-infected cells. Immunofluorescence and transmission electron microscopy indicate that GCRV viroplasms are membraneless structures. Live-cell imaging and fluorescence recovery after photobleaching assay reveal that GCRV viroplasms exhibit liquid-like properties and are highly dynamic structures undergoing fusion and fission. Furthermore, by using a reagent to inhibit the LLPS process and constructing an NS80 mutant defective in LLPS, we confirm that the liquid-like properties of viroplasms are essential for recruiting viral dsRNA, viral RdRp, and viral proteins to participate in viral genome replication and virion assembly, as well as for sequestering host antiviral factors for immune evasion. Collectively, our findings provide detailed insights into reovirus viroplasm formation and reveal the specific functions of LLPS during virus infection and immune evasion, identifying potential targets for the prevention and control of this virus. IMPORTANCE: Grass carp reovirus (GCRV) poses a significant threat to the aquaculture industry, particularly in China, where grass carp is a vital commercial fish species. However, detailed information regarding how GCRV viroplasms form and their specific roles in GCRV infection remains largely unknown. We discovered that GCRV viroplasms exhibit liquid-like properties and are formed through a physico-chemical biological phenomenon known as liquid-liquid phase separation (LLPS), primarily driven by the nonstructural protein NS80. Furthermore, we confirmed that the liquid-like properties of viroplasms are essential for virus replication, assembly, and immune evasion. Our study not only contributes to a deeper understanding of GCRV infection but also sheds light on broader aspects of viroplasm biology. Given that viroplasms are a universal feature of reovirus infection, inhibiting LLPS and then blocking viroplasms formation may serve as a potential pan-reovirus inhibition strategy.
Assuntos
Carpas , Evasão da Resposta Imune , Infecções por Reoviridae , Reoviridae , Proteínas não Estruturais Virais , Replicação Viral , Reoviridae/genética , Reoviridae/fisiologia , Animais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Carpas/virologia , Infecções por Reoviridae/virologia , Corpos de Inclusão Viral/metabolismo , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Citoplasma/virologia , Citoplasma/metabolismo , Genoma Viral , Linhagem Celular , RNA Viral/genética , Separação de FasesRESUMO
The multifunctional tegument protein pUL21 of HSV-2 is phosphorylated in infected cells. We have identified two residues in the unstructured linker region of pUL21, serine 251 and serine 253, as phosphorylation sites. Both phosphorylation sites are absent in HSV-1 pUL21, which likely explains why phosphorylated pUL21 was not detected in cells infected with HSV-1. Cells infected with HSV-2 strain 186 viruses deficient in pUL21 phosphorylation exhibited reductions in both cell-cell spread of virus infection and virus replication. Defects in secondary envelopment of cytoplasmic nucleocapsids were also observed in cells infected with viruses deficient in pUL21 phosphorylation as well as in cells infected with multiple strains of HSV-2 and HSV-1 deleted for pUL21. These results confirm a role for HSV pUL21 in the secondary envelopment of cytoplasmic nucleocapsids and indicate that phosphorylation of HSV-2 pUL21 is required for this activity. Phosphorylation of pUL21 was substantially reduced in cells infected with HSV-2 strain 186 mutants lacking the viral serine/threonine kinase pUL13, indicating a requirement for pUL13 in pUL21 phosphorylation. IMPORTANCE: It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species.
Assuntos
Herpesvirus Humano 2 , Nucleocapsídeo , Replicação Viral , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/fisiologia , Fosforilação , Animais , Chlorocebus aethiops , Humanos , Células Vero , Nucleocapsídeo/metabolismo , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Citoplasma/metabolismo , Citoplasma/virologia , Linhagem Celular , Proteínas Estruturais Virais/metabolismo , Proteínas Estruturais Virais/genética , Montagem de Vírus , Herpes Simples/virologia , Herpes Simples/metabolismoRESUMO
Cytoplasmic male sterility (CMS) arises from the incompatibility between the nucleus and cytoplasm as typical representatives of the chimeric structures in the mitochondrial genome (mitogenome), which has been extensively applied for hybrid seed production in various crops. The frequent occurrence of chimeric mitochondrial genes leading to CMS is consistent with the mitochondrial DNA (mtDNA) evolution. The sequence conservation resulting from faithfully maternal inheritance and the chimeric structure caused by frequent sequence recombination have been defined as two major features of the mitogenome. However, when and how these chimeric mitochondrial genes appear in the context of the highly conserved reproduction of mitochondria is an enigma. This review, therefore, presents the critical view of the research on CMS in plants to elucidate the mechanisms of this phenomenon. Generally, distant hybridization is the main mechanism to generate an original CMS source in natural populations and in breeding. Mitochondria and mitogenomes show pleomorphic and dynamic changes at key stages of the life cycle. The promitochondria in dry seeds develop into fully functioning mitochondria during seed imbibition, followed by massive mitochondria or mitogenome fusion and fission in the germination stage along with changes in the mtDNA structure and quantity. The mitogenome stability is controlled by nuclear loci, such as the nuclear gene Msh1. Its suppression leads to the rearrangement of mtDNA and the production of heritable CMS genes. An abundant recombination of mtDNA is also often found in distant hybrids and somatic/cybrid hybrids. Since mtDNA recombination is ubiquitous in distant hybridization, we put forward a hypothesis that the original CMS genes originated from mtDNA recombination during the germination of the hybrid seeds produced from distant hybridizations to solve the nucleo-cytoplasmic incompatibility resulting from the allogenic nuclear genome during seed germination.
Assuntos
Produtos Agrícolas , DNA Mitocondrial , Genoma Mitocondrial , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , DNA Mitocondrial/genética , Infertilidade das Plantas/genética , Citoplasma/genética , Citoplasma/metabolismo , Melhoramento Vegetal/métodos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Genes MitocondriaisRESUMO
Cytoplasmic male sterility has been a popular genetic tool in development of hybrids. The molecular mechanism behind maternal sterility varies from crop to crop. An understanding of underlying mechanism can help in development of new functional CMS gene in crops which lack effective and stable CMS systems. In crops where seed or fruit is the commercial product, fertility must be recovered in F1 hybrids so that higher yield gains can be realized. This necessitates the presence of fertility restorer gene (Rf) in nucleus of male parent to overcome the effect of sterile cytoplasm. Fertility restoring genes have been identified in crops like wheat, maize, sunflower, rice, pepper, sugar beet, pigeon pea etc. But in crops like eggplant, bell pepper, barley etc. unstable fertility restorers hamper the use of Cytoplasmic genic male sterility (CGMS) system. Stability of CGMS system is influenced by environment, genetic background or interaction of these factors. This review thus aims to understand the genetic mechanisms controlling mitochondrial-nuclear interactions required to design strong and stable restorers without any pleiotropic effects in F1 hybrids.
Assuntos
Citoplasma , Fertilidade , Infertilidade das Plantas , Infertilidade das Plantas/genética , Citoplasma/metabolismo , Citoplasma/genética , Fertilidade/genética , Produtos Agrícolas/genética , Melhoramento Vegetal/métodos , Regulação da Expressão Gênica de Plantas/genética , Núcleo Celular/metabolismo , Núcleo Celular/genéticaRESUMO
Two-pore domain K+ (K2P) channel activity was previously thought to be controlled primarily via a selectivity filter (SF) gate. However, recent crystal structures of TASK-1 and TASK-2 revealed a lower gate at the cytoplasmic pore entrance. Here, we report functional evidence of such a lower gate in the K2P channel K2P17.1 (TALK-2, TASK-4). We identified compounds (drugs and lipids) and mutations that opened the lower gate allowing the fast modification of pore cysteine residues. Surprisingly, stimuli that directly target the SF gate (i.e., pHe., Rb+ permeation, membrane depolarization) also opened the cytoplasmic gate. Reciprocally, opening of the lower gate reduced the electric work to open the SF via voltage driven ion binding. Therefore, it appears that the SF is so rigidly locked into the TALK-2 protein structure that changes in ion occupancy can pry open a distant lower gate and, vice versa, opening of the lower gate concurrently promote SF gate opening. This concept might extent to other K+ channels that contain two gates (e.g., voltage-gated K+ channels) for which such a positive gate coupling has been suggested, but so far not directly demonstrated.
Assuntos
Ativação do Canal Iônico , Canais de Potássio de Domínios Poros em Tandem , Animais , Humanos , Citoplasma/metabolismo , Células HEK293 , Íons/metabolismo , Mutação , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Xenopus laevisRESUMO
Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are core-shell-type membrane-less organelles typically found in the nucleus of mammalian somatic cells but are absent in mouse oocytes. Here, we deliberately induced the assembly of PML-NBs by injecting mRNA encoding human PML protein (hPML VI -sfGFP) into oocytes and investigated their impact on fertilization in which oocyte/embryos undergo multiple types of stresses. Following nuclear membrane breakdown, preassembled hPML VI -sfGFP mRNA-derived PML-NBs (hmdPML-NBs) persisted in the cytoplasm of oocytes, forming less-soluble debris, particularly under stress. Parthenogenetic embryos that successfully formed pronuclei were capable of removing preassembled hmdPML-NBs from the cytoplasm while forming new hmdPML-NBs in the pronucleus. These observations highlight the beneficial aspect of the PML-NB-free nucleoplasmic environment and suggest that the ability to eliminate unnecessary materials in the cytoplasm of metaphase oocytes serves as a potential indicator of the oocyte quality.