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RNA-binding proteins (RBPs) with intrinsically disordered regions (IDRs) are linked to multiple human disorders, but their mechanisms of action remain unclear. Here, we report that one such protein, Nocte, is essential for Drosophila eye development by regulating a critical gene expression cascade at translational level. Knockout of nocte in flies leads to lethality, and its eye-specific depletion impairs eye size and morphology. Nocte preferentially enhances translation of mRNAs with long upstream open reading frames (uORFs). One of the key Nocte targets, glass mRNA, encodes a transcription factor critical for differentiation of photoreceptor neurons and accessory cells, and re-expression of Glass largely rescued the eye defects caused by Nocte depletion. Mechanistically, Nocte counteracts long uORF-mediated translational suppression by promoting translation reinitiation downstream of the uORF. Nocte interacts with translation factors eIF3 and Rack1 through its BAT2 domain, and a Nocte mutant lacking this domain fails to promote translation of glass mRNA. Notably, de novo mutations of human orthologs of Nocte have been detected in schizophrenia patients. Our data suggest that Nocte family of proteins can promote translation reinitiation to overcome long uORFs-mediated translational suppression, and disruption of this function can lead to developmental defects and neurological disorders.
Assuntos
Drosophila , Proteínas de Ligação a RNA , Animais , Humanos , Regiões 5' não Traduzidas , Drosophila/genética , Drosophila/metabolismo , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Topoisomerases are required to release topological stress generated by RNA polymerase II (RNAPII) during transcription. Here, we show that in response to starvation, the complex of topoisomerase 3b (TOP3B) and TDRD3 can enhance not only transcriptional activation, but also repression, which mimics other topoisomerases that can also alter transcription in both directions. The genes enhanced by TOP3B-TDRD3 are enriched with long and highly-expressed ones, which are also preferentially stimulated by other topoisomerases, suggesting that different topoisomerases may recognize their targets through a similar mechanism. Specifically, human HCT116 cells individually inactivated for TOP3B, TDRD3 or TOP3B topoisomerase activity, exhibit similarly disrupted transcription for both starvation-activated genes (SAGs) and starvation-repressed genes (SRGs). Responding to starvation, both TOP3B-TDRD3 and the elongating form of RNAPII exhibit concomitantly increased binding to TOP3B-dependent SAGs, at binding sites that overlap. Notably, TOP3B inactivation decreases the binding of elongating RNAPII to TOP3B-dependent SAGs while increased it to SRGs. Furthermore, TOP3B-ablated cells display reduced transcription of several autophagy-associated genes and autophagy per se. Our data suggest that TOP3B-TDRD3 can promote both transcriptional activation and repression by regulating RNAPII distribution. In addition, the findings that it can facilitate autophagy may account for the shortened lifespan of Top3b-KO mice.
Assuntos
DNA Topoisomerases , Ativação Transcricional , Animais , Humanos , Camundongos , Proteínas/metabolismo , RNA Polimerase II/metabolismo , Linhagem Celular , Fenômenos Fisiológicos Celulares , DNA Topoisomerases/metabolismo , AutofagiaRESUMO
Topoisomerase 3ß (TOP3B) and TDRD3 form a dual-activity topoisomerase complex that interacts with FMRP and can change the topology of both DNA and RNA. Here, we investigated the post-transcriptional influence of TOP3B and associated proteins on mRNA translation and turnover. First, we discovered that in human HCT116 colon cancer cells, knock-out (KO) of TOP3B had similar effects on mRNA turnover and translation as did TDRD3-KO, while FMRP-KO resulted in rather distinct effects, indicating that TOP3B had stronger coordination with TDRD3 than FMRP in mRNA regulation. Second, we identified TOP3B-bound mRNAs in HCT116 cells; we found that while TOP3B did not directly influence the stability or translation of most TOP3B target mRNAs, it stabilized a subset of target mRNAs but had a more complex effect on translation-enhancing for some mRNAs whereas reducing for others. Interestingly, a point mutation that specifically disrupted TOP3B catalytic activity only partially recapitulated the effects of TOP3B-KO on mRNA stability and translation, suggesting that the impact of TOP3B on target mRNAs is partly linked to its ability to change topology of mRNAs. Collectively, our data suggest that TOP3B-TDRD3 can regulate mRNA translation and turnover by mechanisms that are dependent and independent of topoisomerase activity.
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Biossíntese de Proteínas , Proteínas , Humanos , RNA Mensageiro/genéticaRESUMO
Mesoporous Co3O4 (meso-Co3O4)-supported Pt (0.53 wt.% Pt/meso-Co3O4) was synthesized via the KIT-6-templating and polyvinyl alcohol (PVA)-assisted reduction routes. Mesoporous CoO (meso-CoO) was fabricated through in situ reduction of meso-Co3O4 with glycerol, and the 0.18-0.69 wt.% Pt/meso-CoO samples were generated by the PVA-assisted reduction method. Meso-Co3O4 and meso-CoO were of cubic crystal structure and the Pt nanoparticles (NPs) with a uniform size of ca. 2 nm were well distributed on the meso-Co3O4 or meso-CoO surface. The 0.56 wt% Pt/meso-CoO (0.56Pt/meso-CoO) sample performed the best in benzene combustion (T50% = 156 °C and T90% = 186 °C at a space velocity of 80,000 mL/(g h)). Introducing water vapor or CO2 with a certain concentration led to partial deactivation of 0.56 Pt/meso-CoO and such a deactivation was reversible. We think that the superior catalytic activity of 0.56 Pt/meso-CoO was intimately related to its good oxygen activation and benzene adsorption ability.
Assuntos
Benzeno/química , Cobalto , Nanopartículas Metálicas , Modelos Químicos , Platina , Estresse OxidativoRESUMO
Human cells contain five topoisomerases in the nucleus and cytoplasm, but which one is the major topoisomerase for mRNAs is unclear. To date, Top3ß is the only known topoisomerase that possesses RNA topoisomerase activity, binds mRNA translation machinery and interacts with an RNA-binding protein, FMRP, to promote synapse formation; and Top3ß gene deletion has been linked to schizophrenia. Here, we show that Top3ß is also the most abundant mRNA-binding topoisomerase in cells. Top3ß, but not other topoisomerases, contains a distinctive RNA-binding domain; and deletion of this domain diminishes the amount of Top3ß that associates with mRNAs, indicating that Top3ß is specifically targeted to mRNAs by its RNA binding domain. Moreover, Top3ß mutants lacking either its RNA-binding domain or catalytic residue fail to promote synapse formation, suggesting that Top3ß requires both its mRNA-binding and catalytic activity to facilitate neurodevelopment. Notably, Top3ß proteins bearing point mutations from schizophrenia and autism individuals are defective in association with FMRP; whereas one of the mutants is also deficient in binding mRNAs, catalyzing RNA topoisomerase reaction, and promoting synapse formation. Our data suggest that Top3ß is the major topoisomerase for mRNAs, and requires both RNA binding and catalytic activity to promote neurodevelopment and prevent mental dysfunction.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , RNA Mensageiro/metabolismo , Sinapses/fisiologia , Animais , Transtorno Autístico/genética , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Células HEK293 , Humanos , Junção Neuromuscular/crescimento & desenvolvimento , Mutação Puntual , Domínios Proteicos , Esquizofrenia/genéticaRESUMO
FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA Helicases/genética , Humanos , Modelos Moleculares , Mutação , Estresse Fisiológico , Thermoplasma/enzimologiaRESUMO
DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3ß differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3ß proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3ß-polyribosome association requires TDRD3, which directly interacts with Top3ß and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.
Assuntos
DNA Topoisomerases Tipo I/genética , Evolução Molecular , Polirribossomos/genética , Proteínas/genética , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , DNA Super-Helicoidal/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , RNA/genética , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Based on the idea that a layer of properly designed and manufactured fibrous material can serve as both a sound absorber and a structural damper, an approach is introduced in this article to design the microstructure of a fibrous layer for it to provide optimal damping performance. To begin with, previous work related to structural damping by fibrous layers was reviewed. Then the most appropriate models from the literature were adapted for use in the current study. A system comprising an aluminum panel and a limp fibrous layer was driven by a line force, and both the spatial velocity response of the panel and the power distribution within the system were calculated to quantify the degree of damping provided by the fibrous layer, which helped identify the airflow resistivity that provides optimal damping for any frequency and panel of interest. Given the optimal airflow resistivity, a model relating fiber microstructure to airflow resistivity was used to infer the optimal microstructure required to achieve the optimal damping performance. One finding of the current work, for example, was that as the target frequency was reduced, the fiber size required to achieve the optimal damping performance increased.
RESUMO
The RMI subcomplex (RMI1/RMI2) functions with the BLM helicase and topoisomerase IIIα in a complex called the "dissolvasome," which separates double-Holliday junction DNA structures that can arise during DNA repair. This activity suppresses potentially harmful sister chromatid exchange (SCE) events in wild-type cells but not in cells derived from Bloom syndrome patients with inactivating BLM mutations. The RMI subcomplex also associates with FANCM, a component of the Fanconi anemia (FA) core complex that is important for repair of stalled DNA replication forks. The RMI/FANCM interface appears to help coordinate dissolvasome and FA core complex activities, but its precise role remains poorly understood. Here, we define the structure of the RMI/FANCM interface and investigate its roles in coordinating cellular DNA-repair activities. The X-ray crystal structure of the RMI core complex bound to a well-conserved peptide from FANCM shows that FANCM binds to both RMI proteins through a hydrophobic "knobs-into-holes" packing arrangement. The RMI/FANCM interface is shown to be critical for interaction between the components of the dissolvasome and the FA core complex. FANCM variants that substitute alanine for key interface residues strongly destabilize the complex in solution and lead to increased SCE levels in cells that are similar to those observed in blm- or fancm-deficient cells. This study provides a molecular view of the RMI/FANCM complex and highlights a key interface utilized in coordinating the activities of two critical eukaryotic DNA-damage repair machines.
Assuntos
Síndrome de Bloom/metabolismo , DNA Helicases/metabolismo , Alanina/genética , Cristalografia por Raios X/métodos , DNA/genética , Reparo do DNA , Humanos , Modelos Biológicos , Modelos Genéticos , Modelos Moleculares , Conformação Molecular , Estrutura Terciária de Proteína , Recombinação Genética , Reprodutibilidade dos Testes , Troca de Cromátide IrmãRESUMO
Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.
Assuntos
Archaea/química , DNA Helicases/genética , Reparo do DNA , Anemia de Fanconi/genética , Hemaglutininas Virais/genética , Ligases/genética , Proteínas Virais de Fusão/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Evolução Biológica , DNA/metabolismo , DNA Helicases/deficiência , DNA Helicases/metabolismo , Anemia de Fanconi/enzimologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Humanos , Imunoprecipitação , Ligases/deficiência , Ligases/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Transporte Proteico , Ubiquitina/metabolismo , Proteínas Virais de Fusão/deficiênciaRESUMO
The magnetic properties of soft magnetic materials, including their saturation magnetic induction strength and permeability, significantly affect the dynamic characteristics of electromagnetic relays. However, the soft materials most commonly used for relays in the magnetic conductive components of electromagnetic systems, such as electrical pure iron, limit further relay design improvement and optimization to a certain extent. Thus, this paper proposes the use of amorphous and nanocrystalline soft magnetic materials with good high-frequency magnetic properties in magnetic circuits. A wavelet analysis was conducted on the high-frequency components of the coil current while the relay operated, and the corresponding magnetic materials were selected. Considering the challenges in processing amorphous and nanocrystalline materials and collecting test data for the accuracy verification of simulation methods, we prepared a scaled-up prototype for use in dynamic characteristic tests. The simulation method was improved, yielding more accurate simulation results regarding the relay's dynamic characteristics. On this basis, six replacement schemes using amorphous and nanocrystalline materials were considered. The test results proved that this application could improve the relay's dynamic characteristics. Finally, a full-size sample with an iron core consisting of nanocrystalline alloy 1K107B was prepared, and the conclusions were verified in tests.
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Actinomycetes have long been recognized as important sources of clinical antibiotics. However, the exploration of rare actinomycetes, despite their potential for producing bioactive molecules, has remained relatively limited compared to the extensively studied Streptomyces genus. The extensive investigation of Streptomyces species and their natural products has led to a diminished probability of discovering novel bioactive compounds from this group. Consequently, our research focus has shifted towards less explored actinomycetes, beyond Streptomyces, with particular emphasis on Kitasatospora setae (K. setae). The genome of K. setae was annotated and analyzed through whole-genome sequencing using multiple bio-informatics tools, revealing an 8.6 Mbp genome with a 74.42% G + C content. AntiSMASH analysis identified 40 putative biosynthetic gene clusters (BGCs), approximately half of which were recessive and unknown. Additionally, metabolomic mining utilizing mass spectrometry demonstrated the potential for this rare actinomycete to generate numerous bioactive compounds such as glycosides and macrolides, with bafilomycin being the major compound produced. Collectively, genomics- and metabolomics-based techniques confirmed K. setae's potential as a bioactive secondary metabolite producer that is worthy of further exploration.
RESUMO
Evaporation of sweat on the skin surface is the major mechanism for dissipating heat in humans. The secretory capacity of sweat glands (SWGs) declines during aging, leading to heat intolerance in the elderly, but the mechanisms responsible for this decline are poorly understood. We investigated the molecular changes accompanying SWG aging in mice, where sweat tests confirmed a significant reduction of active SWGs in old mice relative to young mice. We first identified SWG-enriched mRNAs by comparing the skin transcriptome of Eda mutant Tabby male mice, which lack SWGs, with that of wild-type control mice by RNA-sequencing analysis. This comparison revealed 171 mRNAs enriched in SWGs, including 47 mRNAs encoding 'core secretory' proteins such as transcription factors, ion channels, ion transporters, and trans-synaptic signaling proteins. Among these, 28 SWG-enriched mRNAs showed significantly altered abundance in the aged male footpad skin, and 11 of them, including Foxa1, Best2, Chrm3, and Foxc1 mRNAs, were found in the 'core secretory' category. Consistent with the changes in mRNA expression levels, immunohistology revealed that higher numbers of secretory cells from old SWGs express the transcription factor FOXC1, the protein product of Foxc1 mRNA. In sum, our study identified mRNAs enriched in SWGs, including those that encode core secretory proteins, and altered abundance of these mRNAs and proteins with aging in mouse SWGs.
Assuntos
Envelhecimento , Glândulas Sudoríparas , Animais , Glândulas Sudoríparas/metabolismo , Camundongos , Envelhecimento/genética , Envelhecimento/metabolismo , Masculino , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , TranscriptomaRESUMO
The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex that can alter both DNA and RNA topology in animals. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impaired cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal short-term memory and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we generated a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, olfaction, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive and psychiatric disorders.
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Disfunção Cognitiva , Regulação da Expressão Gênica , Animais , Humanos , Camundongos , Sequência de Aminoácidos , Neurogênese/genética , Plasticidade Neuronal/genética , Proteínas/genética , Proteínas/metabolismoRESUMO
Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.
Assuntos
Cromossomos Humanos X , Anemia de Fanconi/genética , Metilação de DNA , Mecanismo Genético de Compensação de Dose , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Feminino , Teste de Complementação Genética , Ligação Genética , Humanos , Masculino , Mutação , Proteínas Nucleares/metabolismo , Linhagem , Receptores Androgênicos/metabolismoRESUMO
Risk-stratification of acute myeloid leukemia (AML) based on (cyto)genetic aberrations, including hotspot mutations, deletions and point mutations have evolved substantially in recent years. With the development of next-generation sequence technology, more and more novel mutations in the AML were identified. Thus, to unravel roles and mechanism of novel mutations would improve prognostic and predictive abilities. In this study, two novel germline JAK2 His608Tyr (H608Y) and His608Asn (H608N) mutations were identified and the molecular basis of these mutations in the leukemiagenesis of AML was elucidated. Our results indicated that JAK2 H608Y and H608N mutations disrupted the hydrogen bond between Q656 and H608 which reduced the JH2 domain's activity and abolished interactions between JH1 and JH2 domains, forced JAK2 into the active conformation, facilitated the entrance of substrates and thus caused JAK2 hyperactivation. Further studies suggested that JAK2 H608Y and H608N mutations enhanced the cell proliferation and inhibited the differentiation of Ba/F3 and MV4-11 cells via activating the JAK2-STAT5 signaling pathway. Moreover, rescue experiments demonstrated that mutations repaired the hydrogen bond between Q656 and H608 displayed opposite results. Thus, this study revealed the molecular basis of JAK2 H608Y and H608N mutations in the pathology of AML.
Assuntos
Janus Quinase 2 , Leucemia Mieloide Aguda , Humanos , Janus Quinase 2/metabolismo , Mutação , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proliferação de Células/genética , Transdução de Sinais/genética , Diferenciação CelularRESUMO
The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex in animals that can alter the topology of both DNA and RNA. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impairments in cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal shorten-memory and learning, and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we built a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive impairment and psychiatric disorders.
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Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder with prominent skeletal, renal, immunological, and ectodermal abnormalities. It is caused by mutations of SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA stress response protein. To determine the relationship of this function to the SIOD phenotype, we profiled the cancer prevalence in SIOD and assessed if defects of nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), respectively, explained the ectodermal and immunological features of SIOD. Finally, we determined if Smarcal1(del/del) mice had hypersensitivity to irinotecan (CPT-11), etoposide, and hydroxyurea (HU) and whether exposure to these agents induced features of SIOD. Among 71 SIOD patients, three had non-Hodgkin lymphoma (NHL) and one had osteosarcoma. We did not find evidence of defective NER or NHEJ; however, Smarcal1-deficient mice were hypersensitive to several genotoxic agents. Also, CPT-11, etoposide, and HU caused decreased growth and loss of growth plate chondrocytes. These data, which identify an increased prevalence of NHL in SIOD and confirm hypersensitivity to DNA damaging agents in vivo, provide guidance for the management of SIOD patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DNA Helicases/genética , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/genética , Animais , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , CamundongosRESUMO
A recent study demonstrated that a DNA-RNA dual-activity topoisomerase complex, TOP3B-TDRD3, is required for normal replication of positive-sense RNA viruses, including several human flaviviruses and coronaviruses; and the authors proposed that TOP3B is a target of antiviral drugs. Here we examined this hypothesis by investigating whether inactivation of Top3b can inhibit the replication of a mouse coronavirus, MHV, using cell lines and mice that are inactivated of Top3b or Tdrd3. We found that Top3b-KO or Tdrd3-KO cell lines generated by different CRISPR-CAS9 guide RNAs have variable effects on MHV replication. In addition, we did not find significant changes of MHV replication in brains or lungs in Top3B-KO mice. Moreover, immunostaining showed that Top3b proteins are not co-localized with MHV replication complexes but rather, localized in stress granules in the MHV-infected cells. Our results suggest that Top3b does not have a universal role in promoting replication of positive-sense RNA virus, and cautions should be taken when targeting it to develop anti-viral drugs.
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Infecções por Coronavirus , Coronavirus , Vírus da Hepatite Murina , Vírus de RNA , Animais , Camundongos , Antivirais/farmacologia , Linhagem Celular , Coronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/metabolismo , Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Replicação ViralRESUMO
Objective To investigate the killing effect and molecular mechanism of aberrant expression of calnexin (CNX) in the colorectal cancer (CRC) on the CD8+ T immune cells. Methods Immunohistochemistry was used to detect CNX protein level in 102 pairs of CRC cancer and adjacent non-cancerous tissues. Western blotting was employed to examine the protein expression of MHC I in the HCT-15 cells overexpressed with CNX or in the SW480 cells whose CNX expressions were knockdown by siRNA. Murine CD8+ T cells isolated from the spleen were cocultured with CT-26 murine CRC cells infected with lentivirus-mediated CNX overexpression. The killing effect of CD8+ T cells on CT-26 cells was determined by cytotoxicity kit. The secretion of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) in the culture medium were examined by ELISA. Results The protein level of CNX in colorectal cancer tissues were significantly lower than that in non-cancerous tissues. CNX overexpressed in HCT-15 cells was upregulated and CNX knockdown in SW480 cells downregulated the MHC I expression in these cells. Furthermore, the overexpression of CNX could not only enhance the killing effect of CD8+ T cells on CT-26 cells, but also promote the secretion of IFN-γ and TNF-α from these cells. Conclusion CNX can enhance the killing potential of CD8+ T cells on tumor cells through upregulating the MHC I expression in colorectal cancer cells.