RESUMO
Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.
Assuntos
Corpos de Inclusão Viral , Antígeno-1 Intracelular de Células T , Replicação Viral , eIF-2 Quinase , eIF-2 Quinase/metabolismo , eIF-2 Quinase/genética , Animais , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/genética , Chlorocebus aethiops , Células Vero , Corpos de Inclusão Viral/metabolismo , Humanos , Grânulos de Estresse/metabolismo , Corpos de Inclusão/metabolismo , Interações Hospedeiro-Patógeno , Grânulos Citoplasmáticos/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Separação de FasesRESUMO
Senescent cells exhibit a diverse spectrum of changes in their morphology, proliferative capacity, senescence-associated secretory phenotype (SASP) production, and mitochondrial homeostasis. These cells often manifest with elongated mitochondria, a hallmark of cellular senescence. However, the precise regulatory mechanisms orchestrating this phenomenon remain predominantly unexplored. In this study, we provide compelling evidence for decreases in TIA-1, a pivotal regulator of mitochondrial dynamics, in models of both replicative senescence and ionizing radiation (IR)-induced senescence. The downregulation of TIA-1 was determined to trigger mitochondrial elongation and enhance the expression of senescence-associated ß-galactosidase, a marker of cellular senescence, in human foreskin fibroblast HS27 cells and human keratinocyte HaCaT cells. Conversely, the overexpression of TIA-1 mitigated IR-induced cellular senescence. Notably, we identified the miR-30-5p family as a novel factor regulating TIA-1 expression. Augmented expression of the miR-30-5p family was responsible for driving mitochondrial elongation and promoting cellular senescence in response to IR. Taken together, our findings underscore the significance of the miR-30-5p/TIA-1 axis in governing mitochondrial dynamics and cellular senescence.
Assuntos
Senescência Celular , MicroRNAs , Mitocôndrias , Dinâmica Mitocondrial , Antígeno-1 Intracelular de Células T , Humanos , MicroRNAs/metabolismo , MicroRNAs/genética , Senescência Celular/efeitos da radiação , Senescência Celular/genética , Dinâmica Mitocondrial/genética , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/genética , Mitocôndrias/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Linhagem Celular , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Queratinócitos/citologia , Transdução de Sinais , Radiação IonizanteRESUMO
T-cell intracellular antigen-1 (TIA-1) is a key RNA-binding protein that participates in translation regulation and RNA splicing. TIA-1 undergoes liquid-liquid phase separation as a fundamental mechanism that enables the condensation of RNA and proteins into membraneless organelles called stress granules (SGs). However, this dynamic behavior can lead to aberrant fibril formation, implicated in neurodegenerative disorders, and must be tightly regulated. In this study, we investigated the role in the cell of histidine residues His94 and His96, responsible for Zn2+ binding. Using fluorescence microscopy, we found that the specific binding site formed by these residues is critical for SG assembly. Furthermore, it also plays a role maintaining the dynamic behavior of SG-assembled TIA-1. Collectively, our findings confirm the physiological relevance of TIA-1 His94 and His96 in the Zn2+-mediated regulatory mechanism for protection against fibril formation in SGs.
Assuntos
Histidina , Grânulos de Estresse , Antígeno-1 Intracelular de Células T , Zinco , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/genética , Zinco/metabolismo , Histidina/metabolismo , Histidina/genética , Histidina/química , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genética , Humanos , Ligação Proteica , Sítios de Ligação , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/genéticaRESUMO
Viral RNA-host protein interactions are indispensable during RNA virus transcription and replication, but their detailed structural and dynamical features remain largely elusive. Here, we characterize the binding interface for the SARS-CoV-2 stem-loop 3 (SL3) cis-acting element to human TIA1 protein with a combined theoretical and experimental approaches. The highly structured SARS-CoV-2 SL3 has a high binding affinity to TIA1 protein, in which the aromatic stacking, hydrogen bonds, and hydrophobic interactions collectively direct this specific binding. Further mutagenesis studies validate our proposed 3D binding model and reveal two SL3 variants have enhanced binding affinities to TIA1. And disruptions of the identified RNA-protein interactions with designed antisense oligonucleotides dramatically reduce SARS-CoV-2 infection in cells. Finally, TIA1 protein could interact with conserved SL3 RNA elements within other betacoronavirus lineages. These findings open an avenue to explore the viral RNA-host protein interactions and provide a pioneering structural basis for RNA-targeting antiviral drug design.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , RNA Viral/metabolismo , Ligação Proteica , COVID-19/genética , Mutagênese , Antígeno-1 Intracelular de Células T/metabolismoRESUMO
As the most common subtype of lung cancer, non-small cell lung cancer (NSCLC)is responsible for a large proportion of global cancer-caused deaths. The implication of long non-coding RNAs (lncRNAs) as tumor-suppressor or carcinogenic genes in NSCLC has been widely documented. Our study sought to investigate the performance of lncRNA RAMP2 antisense RNA1 (RAMP2-AS1) in NSCLC. GEPIA bioinformatics tool and RT-qPCR were applied for assessing the expression of RAMP2-AS1 and its neighboring gene receptor activity-modifying protein 2 (RAMP2) in NSCLC. Functional assays including CCK-8 assay, colony formation assay as well as caspase-3 activity analysis and Transwell invasion assays were applied for detecting the biological phenotypes of NSCLC cells. Interaction among RAMP2-AS1, RAMP2 and T-cell intracellular antigen 1cytotoxic granule associated RNA binding protein (TIA1) was evaluated by RNA immunoprecipitation and pulldown assays. We found that RAMP2-AS1 and RAMP2 were downregulated in NSCLC. Overexpression of RAMP2-AS1 hampered proliferation and invasion, whereas induced apoptosis of NSCLC cells. Mechanistically, RAMP2-AS1 interacted with TIA1 to stabilize the mRNA of RAMP2. In conclusion, we first uncovered that RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of NSCLC, providing new insight to improve the treatment efficacy of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , RNA Longo não Codificante , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/genética , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Linhagem Celular Tumoral , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Movimento Celular/genética , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismoRESUMO
B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins T cell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.
Assuntos
Linfopoese , Proteínas de Ligação a Poli(A) , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Linfopoese/genética , Splicing de RNA , Sítios de Splice de RNA , Reparo do DNA , Dano ao DNARESUMO
BACKGROUND: Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer's disease (AD). This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy. METHODS: The primary hippocampal neurons, N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy, which was analysed by Student's two-tailed t-test. The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1 (mTORC1) activity and the vacuolar H+-ATPase (v-ATPase) activity, respectively, which were analysed by One-way ANOVA with post hoc tests. The Western blotting, co-immunoprecipitation and immunofluorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations, as analysed by Student's two-tailed t-test or One-way ANOVA with post hoc tests. The autophagosome formation was detected by immunofluorescence staining and transmission electron microscopy. The amino acids (AA) levels were detected by high performance liquid chromatography (HPLC). RESULTS: We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits. Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1 (PRD-TIA1) and this association significantly increased the intercellular level of amino acids (Leucine, P = 0.0038; Glutamic acid, P = 0.0348; Alanine, P = 0.0037; Glycine, P = 0.0104), with concordant upregulation of mTORC1 activity [phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1), P < 0.0001; phosphorylated 70 kDa ribosomal protein S6 kinase 1 (p-p70S6K1), P = 0.0001, phosphorylated unc-51-like autophagy-activating kinase 1 (p-ULK1), P = 0.0015] and inhibition of autophagosome formation [microtubule-associated protein light chain 3 II (LC3 II), P = 0.0073; LC3 puncta, P < 0.0001]. As expected, this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation. Importantly, we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1, downregulating the endogenous TIA1 expression by shRNA, or downregulating tau protein level by a small proteolysis targeting chimera (PROTAC) could remarkably attenuate tau-induced autophagy impairment. CONCLUSIONS: Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway, and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treatment and that of related tauopathies.
Assuntos
Autofagossomos , Alvo Mecanístico do Complexo 1 de Rapamicina , Antígeno-1 Intracelular de Células T , Proteínas tau , Aminoácidos/metabolismo , Autofagossomos/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Antígeno-1 Intracelular de Células T/metabolismo , Proteínas tau/metabolismo , Proteínas tau/farmacologiaRESUMO
BACKGROUND: CRNDE is known to be an important predictive factor of prognosis in many tumors; however, its role in cisplatin resistance is still unknown in ovarian cancer. The aim of the current research was to investigate the association between CRNDE and cisplatin resistance. MATERIALS AND METHODS: QRT-PCR and in situ hybridization assay were employed to detect the expression of CRNDE in ovarian cancer cells and tissues; CCK8 assay, AnnexinV-FITC apoptosis assay and Trans-well assay, to determine the cell proliferation, apoptosis and invasion; and RNA-pull down assay, mass spectrometry analysis, gene microarray to search the targeted gene of CRNDE and SRSF1. Association of CRNDE with SRSF1 was determined in ovarian cancer cells and nude mice. RESULTS: It was found that CRNDE and SRSF1 expression were higher in the cisplatin resistant ovarian cancer cells than their control cells. High expression of CRNDE and SRSF1 led to cisplatin resistance. While inhibition of CRNDE or SRSF1 sensitized ovarian cancer to cisplatin in vitro and in vivo. Moreover, as indicated in RIP assay, SRSF1 was potentially the targeted gene of CRNDE, and CRNDE promoting SRSF1 expression to induce cisplatin resistance; as indicated in gene microassay, there was significantly positive correlation between SRSF1 and TIA1, and SRSF1 promoting TIA1 expression. CONCLUSION: In conclusion, CRNDE induced cisplatin resistance in ovarian cancer through SRSF1/TIA1 signaling pathway; thus, CRNDE inhibitor or SRSF1 inhibitor combined with cisplatin might act as a novel promising approach to ovarian cancer.
Assuntos
MicroRNAs , Neoplasias Ovarianas , RNA Longo não Codificante/genética , Animais , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Transdução de Sinais , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismoRESUMO
Spinocerebellar ataxia type 7 (SCA7) and other polyglutamine (polyQ) diseases are caused by expansions of polyQ repeats in disease-specific proteins. Aggregation of the polyQ proteins resulting in various forms of cellular stress, that could induce the stress granule (SG) response, is believed to be a common pathological mechanism in these disorders. SGs can contribute to cell survival but have also been suggested to exacerbate disease pathology by seeding protein aggregation. In this study, we show that two SG-related proteins, TDP-43 and TIA1, are sequestered into the aggregates formed by polyQ-expanded ATXN7 in SCA7 cells. Interestingly, mutant ATXN7 also localises to induced SGs, and this association altered the shape of the SGs. In spite of this, neither the ability to induce nor to disassemble SGs, in response to arsenite stress induction or relief, was affected in SCA7 cells. Moreover, we could not observe any change in the number of ATXN7 aggregates per cell following SG induction, although a small, non-significant, increase in total aggregated ATXN7 material could be detected using filter trap. However, mutant ATXN7 expression in itself increased the speckling of the SG-nucleating protein G3BP1 and the SG response. Taken together, our results indicate that the SG response is induced, and although some key modulators of SGs show altered behaviour, the dynamics of SGs appear normal in the presence of mutant ATXN7.
Assuntos
DNA Helicases , Ataxias Espinocerebelares , Ataxina-7/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ataxias Espinocerebelares/genética , Grânulos de Estresse , Antígeno-1 Intracelular de Células T/metabolismoRESUMO
T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is primarily involved in the post-transcriptional regulation of cellular RNAs. Furthermore, it is a key component of stress granules (SGs), RNA, and protein aggregates that are formed in response to stressful stimuli to reduce cellular activity as a survival mechanism. TIA1 p.E384K mutation is the genetic cause of Welander distal myopathy (WDM), a late-onset muscular dystrophy whose pathogenesis has been related to modifying SG dynamics. In this study, we present the results obtained by analyzing two specific aspects: (i) SGs properties and dynamics depending on the amino acid at position 384 of TIA1; and (ii) the formation/disassembly time-course of TIA1WT/WDM-dependent SGs under oxidative stress. The generation of TIA1 variants-in which the amino acid mutated in WDM and the adjacent ones were replaced by lysines, glutamic acids, or alanines-allowed us to verify that the inclusion of a single lysine is necessary and sufficient to alter SGs dynamics. Moreover, time-lapse microscopy analysis allowed us to establish in vivo the dynamics of TIA1WT/WDM-dependent SG formation and disassembly, after the elimination of the oxidizing agent, for 1 and 3 h, respectively. Our observations show distinct dynamics between the formation and disassembly of TIA1WT/WDM-dependent SGs. Taken together, this study has allowed us to expand the existing knowledge on the role of TIA1 and the WDM mutation in SG formation.
Assuntos
Miopatias Distais , Aminoácidos/metabolismo , Miopatias Distais/genética , Miopatias Distais/metabolismo , Humanos , Estresse Oxidativo , Proteostase , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Grânulos de Estresse , Antígeno-1 Intracelular de Células T/metabolismo , Linfócitos T/metabolismoRESUMO
T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is expressed in many tissues and in the vast majority of species, although it was first discovered as a component of human cytotoxic T lymphocytes. TIA1 has a dual localization in the nucleus and cytoplasm, where it plays an important role as a regulator of gene-expression flux. As a multifunctional master modulator, TIA1 controls biological processes relevant to the physiological functioning of the organism and the development and/or progression of several human pathologies. This review summarizes our current knowledge of the molecular aspects and cellular processes involving TIA1, with relevance for human pathophysiology.
Assuntos
Núcleo Celular , Proteínas de Ligação a RNA , Antígeno-1 Intracelular de Células T , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Proteínas de Ligação a RNA/metabolismo , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismo , Linfócitos T/metabolismoRESUMO
Early life radiation exposure causes abnormal brain development, leading to adult depression. However, few studies have been conducted to explore pre- or post-natal irradiation-induced depression-related neuropathological changes. Relevant molecular mechanisms are also poorly understood. We induced adult depression by irradiation of mice at postnatal day 3 (P3) to reveal hippocampal neuropathological changes and investigate their molecular mechanism, focusing on MicroRNA (miR) and its target mRNA and protein. P3 mice were irradiated by γ-rays with 5Gy, and euthanized at 1, 7 and 120 days after irradiation. A behavioral test was conducted before the animals were euthanized at 120 days after irradiation. The animal brains were used for different studies including immunohistochemistry, CAP-miRSeq, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and western blotting. The interaction of miR-34a-5p and its target T-cell intracytoplasmic antigen-1 (Tia1) was confirmed by luciferase reporter assay. Overexpression of Tia1 in a neural stem cell (NSC) model was used to further validate findings from the mouse model. Irradiation with 5 Gy at P3 induced depression in adult mice. Animal hippocampal pathological changes included hypoplasia of the infrapyramidal blade of the stratum granulosum, aberrant and impaired cell division, and neurogenesis in the dentate gyrus. At the molecular level, upregulation of miR-34a-5p and downregulation of Tia1 mRNA were observed in both animal and neural stem cell models. The luciferase reporter assay and gene transfection studies further confirmed a direct interaction between miR-43a-5p and Tia1. Our results indicate that the early life γ-radiation-activated miR-43a-5p/Tia1 pathway is involved in the pathogenesis of adult depression. This novel finding may provide a new therapeutic target by inhibiting the miR-43a-5p/Tia1 pathway to prevent radiation-induced pathogenesis of depression.
Assuntos
Giro Denteado/patologia , Depressão/patologia , Raios gama/efeitos adversos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , MicroRNAs/genética , Neurogênese , Antígeno-1 Intracelular de Células T/metabolismo , Animais , Apoptose , Proliferação de Células , Giro Denteado/efeitos da radiação , Depressão/etiologia , Depressão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Antígeno-1 Intracelular de Células T/genéticaRESUMO
BACKGROUND: Neuronal development is a tightly controlled process involving multi-layered regulatory mechanisms. While transcriptional pathways regulating neurodevelopment are well characterized, post-transcriptional programs are still poorly understood. TIA1 is an RNA-binding protein that can regulate splicing, stability, or translation of target mRNAs, and has been shown to play critical roles in stress response and neurodevelopment. However, the identity of mRNAs regulated by TIA1 during neurodevelopment under unstressed conditions is still unknown. METHODS AND RESULTS: To identify the mRNAs targeted by TIA1 during the first stages of human neurodevelopment, we performed RNA immunoprecipitation-sequencing (RIP-seq) on human embryonic stem cells (hESCs) and derived neural progenitor cells (NPCs), and cortical neurons under unstressed conditions. While there was no change in TIA1 protein levels, the number of TIA1 targeted mRNAs decreased from pluripotent cells to neurons. We identified 2400, 845, and 330 TIA1 mRNA targets in hESCs, NPC, and neurons, respectively. The vast majority of mRNA targets in hESC were genes associated with neurodevelopment and included autism spectrum disorder-risk genes that were not bound in neurons. Additionally, we found that most TIA1 mRNA targets have reduced ribosomal engagement levels. CONCLUSION: Our results reveal TIA1 mRNA targets in hESCs and during human neurodevelopment, indicate that translation repression is a key process targeted by TIA1 binding and implicate TIA1 function in neuronal differentiation.
Assuntos
Neurogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismo , Transtorno do Espectro Autista/genética , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imunoprecipitação/métodos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos , TransfecçãoRESUMO
Recent studies revealed the biological significance of dynamic multicomponent assemblies of biomolecules inside living cells. Protein and nucleic acid assemblies are biomolecular condensates or non-membrane-bound organelles that have attracted increasing attention. Synthetic tools that manipulate the dynamic assembly/disassembly process of the structures are useful in elucidating both biophysical mechanisms of their assembly/disassembly and physiological roles of the condensates. In this report, general protocols to form and observe synthetic polymer-based condensates in living cells are described using the tool iPOLYMER. Taking advantage of the modular design of the tool, both chemical and light stimuli can induce formation of synthetic condensates inside living cells, which are observed by laser-scanning confocal microscopy. The experimental design described herein should help those who conduct experiments on synthetic manipulation of biomolecular condensates using iPOLYMER and other tools for synthetic manipulation of condensates. Technical notes for using iPOLYMER, including basic protocols of chemical- or light-inducible dimerization techniques (CID/LID), choice of proper control experiments, and advantages/disadvantages are also presented.
Assuntos
Engenharia Celular , Grânulos Citoplasmáticos/genética , Regulação da Expressão Gênica , Mimetismo Molecular , Optogenética , Peptídeos/genética , RNA/genética , Biologia Sintética , Antígeno-1 Intracelular de Células T/genética , Animais , Células COS , Técnicas de Cultura de Células , Chlorocebus aethiops , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células HEK293 , Humanos , Hidrogéis , Luz , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/metabolismo , Domínios Proteicos , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia , Antígeno-1 Intracelular de Células T/metabolismo , TransfecçãoRESUMO
T-cell restriction intracellular antigen 1 (TIA1) is an RNA-binding protein that is a major component of stress granules (SGs). The low complexity domain (LCD) of TIA1 plays a central role in facilitating SGs assembly through liquid-liquid phase separation (LLPS). Disruption of the LLPS process has been associated with several diseases. It has recently been shown that the proline-rich domain affects the LLPS process of some proteins (such as UBQLN2 and Tau). Thus, proline may regulate LLPS. The LCD of TIA1 contains 11 proline residues, and several proline-related mutations have been shown to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we demonstrated that TIA1 can undergo phase separation in cells. Additionally, disease-associated proline-to-leucine (P-L) mutations, which altered droplet morphology, facilitated the liquid-to-solid phase transition of TIA1 into solid-like amyloid fibrils. The changes in the physical properties of the P-L mutation altered the behavior of TIA1 in vivo and led to abnormal SGs kinetics, resulting in the formation of the pathological inclusions of ALS. Prolines are the key residues for regulating the LLPS of TIA1.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Agregados Proteicos/genética , Antígeno-1 Intracelular de Células T/genética , Amiloide/genética , Grânulos Citoplasmáticos/fisiologia , Corpos de Inclusão/patologia , Extração Líquido-Líquido , Mutação/genética , Prolina/metabolismo , Agregados Proteicos/fisiologia , Agregação Patológica de Proteínas , Domínios Proteicos/genética , Grânulos de Estresse/genética , Grânulos de Estresse/metabolismo , Antígeno-1 Intracelular de Células T/metabolismoRESUMO
BACKGROUND: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. RESULTS: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. CONCLUSIONS: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.
Assuntos
Processamento Alternativo/genética , Reprogramação Celular/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Antígeno-1 Intracelular de Células T/metabolismo , Animais , Linfócitos B/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.
Assuntos
Agregados Proteicos , Agregação Patológica de Proteínas , Multimerização Proteica , Antígeno-1 Intracelular de Células T/metabolismo , Proteínas tau/metabolismo , Amiloide/química , Amiloide/metabolismo , Humanos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Recombinantes , Proteínas tau/químicaRESUMO
TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3' UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.
Assuntos
RNA/metabolismo , Antígeno-1 Intracelular de Células T/química , Regiões 3' não Traduzidas , Amiloide/ultraestrutura , Sítios de Ligação , DNA/química , DNA/metabolismo , Humanos , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Conformação Proteica , RNA/química , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/ultraestrutura , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Kawasaki disease (KD) is a systemic vasculitis of unknown etiology which predominantly affects medium- and small-sized muscular arteries. Histopathologic studies of KD vasculitis lesions have demonstrated characteristic T cell infiltration and an abundance of CD8 T cells; however, the contribution of cytotoxic lymphocytes to KD vasculitis lesions has not been identified. Here, we histopathologically and immunohistochemically examined infiltrating inflammatory cells, particularly cytotoxic protein-positive cells, such as granzyme B cells and TIA-1 cells, in KD vasculitis lesions. Three autopsy specimens with acute-phase KD were observed and contained 24 vasculitis lesions affecting medium-sized muscular arteries, excluding pulmonary arteries. Infiltrating neutrophils in vasculitis lesions were evaluated by hematoxylin and eosin staining, and monocytes/macrophages and lymphocytes were evaluated by immunohistochemistry. The predominant cells were CD163 monocytes/macrophages and CD3 T cells. CD8 T cells, granzyme B cells, and TIA-1 cells were also observed, but CD56 natural killer cells were rare. To the best of our knowledge, the current study is the first histopathologic report confirming the infiltration of inflammatory cells with cytotoxic proteins in vasculitis lesions in patients with KD. Cytotoxic T cells may play a role in the development of vasculitis lesions in KD patients.
Assuntos
Arterite/metabolismo , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Síndrome de Linfonodos Mucocutâneos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Arterite/patologia , Antígeno CD56/metabolismo , Feminino , Granzimas/metabolismo , Humanos , Lactente , Células Matadoras Naturais/patologia , Macrófagos/patologia , Masculino , Síndrome de Linfonodos Mucocutâneos/patologia , Antígeno-1 Intracelular de Células T/metabolismo , Linfócitos T Citotóxicos/patologiaRESUMO
Stress granules (SGs) are stress-induced RNA-protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple elements. Moreover, artificial tethering of G3BP1, TIA1, or FMRP can target mRNAs into SGs in a dose-dependent manner with numerous interactions required for efficient SG partitioning, which suggests individual protein interactions have small effects on the SG partitioning of mRNPs. This is supported by the observation that the SG transcriptome is largely unchanged in cell lines lacking the abundant SG RNA-binding proteins G3BP1 and G3BP2. We suggest the targeting of RNPs into SGs is due to a summation of potential RNA-protein, protein-protein, and RNA-RNA interactions with no single interaction dominating RNP recruitment into SGs.