An RNA topoisomerase complex interacts with Fragile X syndrome protein to promote neurodevelopment and maintain normal life-span

RNA 拓扑异构酶复合物与脆性 X 综合征蛋白相互作用，促进神经发育并维持正常寿命

• 批准号: 10467897
• 负责人: Weidong Wang
• 金额: $ 5万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467897
• 关键词: Adult; Affect; Animals; Anxiety; Archaea; Bacteria; Binding; Biochemical; Biogenesis; Biological Assay; Brain; CRISPR/Cas technology; Cell Nucleus; Cells; Collaborations; Communication; Complex; Cytoplasm; DNA; DNA Repair; DNA Topoisomerases; DNA Transposable Elements; DNA metabolism; Data; Defect; Development; Disease; Drosophila genus; Enzymes; Eukaryota; Evolution; Exhibits; Eye; FMR1; FMRP; Focal Adhesion Kinase 1; Fragile X Syndrome; Fright; Functional disorder; Gene Silencing; Genes; Genetic Transcription; Genetic Translation; Genetic study; Goals; Heterochromatin; Hippocampus (Brain); Human; Human Genetics; Immunoprecipitation; Impaired cognition; Impairment; Intellectual functioning disability; Journals; Knock-out; Knockout Mice; Learning; Life; Ligase; Link; Longevity; Manuscripts; Memory; Mental Health; Mental disorders; Messenger RNA; Metabolism; Modeling; Mus; Mutant Strains Mice; Mutation; Nature; Neurology; Neuromuscular Junction; Neurons; Neurosciences; Oocytes; PTK2 gene; Pathogenesis; Pathway interactions; Patients; Penetrance; Pharmaceutical Preparations; Phenotype; Point Mutation; Polymerase; Polyribosomes; Process; Protein Tyrosine Kinase; Proteins; Psyche structure; Publishing; RNA; RNA Binding; RNA Interference; RNA Recognition Motif; RNA metabolism; Reaction; Reporting; Research; Schizophrenia; Single Nucleotide Polymorphism; Small Interfering RNA; Social Interaction; Stress; Synapses; Synaptic Transmission; Synaptic plasticity; System; Testing; Topoisomerase; Transgenic Organisms; Work; Yeasts; autism spectrum disorder; behavioral phenotyping; conditioned fear; crosslink; crosslinking and immunoprecipitation sequencing; dopaminergic neuron; fly; helicase; inhibitor/antagonist; mRNA Expression; mutant; neurodevelopment; neurogenesis; nuclease; overexpression; paralogous gene; protein p68; repaired; response; stress granule; synaptogenesis

Topoisomerases are magicians of the DNA world, working their wizardry to solve topological problems of DNA during replication, repair, and transcription. Many DNA metabolizing enzymes (polymerases, helicases, nucleases, and ligases) have counterparts in the RNA world. One exception is topoisomerase, which seems to be absent from the RNA world. During our research on DNA topoisomerases that participate in DNA repair, we discovered that topoisomerase 3beta (Top3b) has many features of an RNA topoisomerase. First, Top3b associates with the Fragile X syndrome protein, FMRP, which is known to bind mRNA and to regulate mRNA translation and transport. Second, Top3b resembles FMRP in associating with polyribosomes, which are units for mRNA translation. Third, Top3b colocalizes with FMRP in RNA stress granules, which are cytoplasmic compartments for stalled mRNA and translation machinery. Fourth, Top3b binds mRNA in cells as shown by a crosslinked-RNA immunoprecipitation assay (HITS-CLIP). Fourth, Top3b mutants in Drosophila display abnormal neuromuscular junctions similar to those in FMR1 mutants. Fifth, Top3b mutations in Drosophila modify the rough eye phenotype induced by FMRP over-expression. Sixth and most importantly, Top3b can directly catalyze topoisomerase reactions on RNA substrates. In addition, a point mutation that inactivates its DNA topoisomerase activity also disrupts its RNA topoisomerase activity, indicating that the same catalytic residue may be used for reactions on both DNA and RNA substrates. Furthermore, the paralog of Top3b, Top3a, completely lacks RNA topoisomerase activity, suggesting that the observed RNA topoisomerase activity is specific for Top3b. Recently, we were able to create Drosophila Top3b/Fmr1 double mutant, and found that the abnormal neuromuscular junction phenotype observed in each single mutant is suppressed in the double mutant. This further illustrates that the two proteins genetically interact in antagonistic manner. Moreover, the data suggest that the inhibitors of the RNA topoisomerase may be used as drugs to alleviate conditions of the Fragile X patients. Recent human studies have linked Top3b mutation with schizophrenia, intellectual disability and autism. Consistent with these findings, we found that Top3b bound multiple mRNAs that are encoded by schizophrenia and autism-related genes. We further showed that one schizophrenia-related gene, ptk2/FAK, displayed reduced expression in neuromuscular junctions of the Drosophila Top3b mutant, Fmr1 mutant, and their double mutant, suggesting that Top3b and Fmr1 work in the same pathway to promote ptk2 expression in synapse. We also observed abnormal synapse formation in both Drosophila and mouse that are inactivated of Top3b. In summary, we have identified Top3b as the first dual-activity topoisomerase in eukaryotes that can act on not only DNA, but also RNA. We showed that Top3b works with FMRP to promote neurodevelopment and mental health. A manuscript describing this work has been published in Nature Neuroscience (Xu et al. Nature Neuroscience, 2013), and is featured by highlights in Nature, Nature Neuroscience, Nature Review Neurology, and other journals and organizations. One important issue is how prevalent is RNA topoisomerase activity in various species. We have tested topoisomerases from a variety of species, and found that RNA topoisomerase activity is present in all three domains of life, bacteria, archaea, and eukarya. The data support the notion that the RNA topoisomerases are important so that they are conserved through evolution. We and others have previously shown that Top3b in human associate with polyribosomes, suggesting that it participates in mRNA translation. We have now found that Top3b from other animal species also associate with polyribosomes, whereas those from bacteria and yeast do not. Moreover, this association requires TDRD3, a partner of Top3b which only exists in animals. Our data thus suggest that only in animals, the dual-activity topoisomerase Top3b works in mRNA translation as part of a complex. Human cells have 5 topoisomerases in nucleus and cytoplasm. We found that only Top3b strongly binds mRNAs. Moreover, we found that this binding activity strongly depends on the RNA-binding domain of Top3b. These data are consistent with the notion that Top3b acts as an RNA topoisomerase and works in mRNA metabolism. We have examined two de novo single nucleotide variants of Top3b discovered in schizophrenia and autism patients, and found that an autism patient-derived point mutant lost the RNA topoisomerase activity and mRNA binding activity. In addition, both mutants have defective ability to interact with FMRP The data provide additional evidence for involvement of Top3b in mental disorders. We produced transgenic flies expressing different mutants of Top3b. We found that the RNA binding activity and the topoisomerase activity are both required for formation of normal Drosophila synapase. Moreover, the autism patient derived point mutant is defective in promoting synapse formation. The data support the notion that the RNA topoiomerase activity of Top3b is needed for normal neurodevelopment. We have shown that Top3-knockout mice exhibit behavioral phenotypes related to psychiatric disorders and cognitive impairment, including increased anxiety and fear, impaired social interactions, and defective spatial learning and memory. In addition, these mice display deficits in adult hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many that are critical for neuronal functions and mental health. Our data suggest that Top3 is essential for normal brain function in multiple domains, and defective neuronal activity-dependent transcription may be a mechanism by which Top3 mutations cause cognitive impairment and psychiatric disorders. This work is under revision in Nature Communications. We found that TDRD3 forms a stable complex with Top3b in not only human, but also Drosophila. We show that in Drosophila, Top3b biochemically and genetically interacts with the RNAi-induced silencing complex (RISC) containing AGO2, p68 RNA helicase, and FMRP. Top3b and RISC mutants are both defective in heterochromatin formation and transcriptional silencing; and this defect is suppressed in the double mutants between Top3b and AGO2, p68, and RNAi biogenesis enzyme, Dicer-2, suggesting coordinated interactions between Top3b and siRNA machinery. Moreover, both Top3b and AGO2 single mutant flies exhibit reduced heterochromatin markers in pericentric and telomeric heterochromatin; and the reduction in pericentric heterochromatin is also suppressed in their double mutant. Furthermore, expression of several genes and transposable elements within telomeric heterochromatin is increased in the Top3b mutant. Finally, we found that Top3b depends on its RNA binding domain and its catalytic activity to promote heterochromatin formation and transcriptional silencing. Our data suggest that Top3b works with siRNA machinery to promote heterochromatin formation and transcriptional silencing. This work is in Nature Communications. We have established CRISPR-CAS9 system in the lab, and were able to knockout TDRD3 in Drosophila. We found that TDRD3 suppresses Top3b function, but works cooperatively with Fmr1. TDRD3 flies also display abnormal development of DA neurons. Their oocyte development is also defective. We are now investigating the underlying mechanism. We have established a TDRD3 mutant mouse line. In collaboration with Yue Wang in Mark Mattson's lab, we found that the mutant line has defective synaptic transmission. We also observed high penetrance of lethality.

拓扑异构酶是DNA世界的魔术师，在复制，修复和转录过程中努力解决DNA的拓扑问题。许多DNA代谢酶（聚合酶，解旋酶，核酸酶和连接酶）在RNA世界中具有对应物。拓扑异构酶是一个例外，RNA世界似乎不存在。 在参与DNA修复的DNA拓扑异构酶的研究中，我们发现拓扑异构酶3Beta（Top3b）具有RNA拓扑异构酶的许多特征。首先，Top3b与脆弱的X综合征蛋白FMRP相关联，该综合征蛋白FMRP已知可以结合mRNA并调节mRNA翻译和运输。其次，TOP3B与fMRP与多核糖体关联，这是mRNA翻译的单位。第三，TOP3B与RNA应力颗粒中的FMRP共定位，它们是停滞mRNA和翻译机械的细胞质室。第四，TOP3B结合细胞中的mRNA，如交联-RNA免疫沉淀测定法（hits-CLIP）所示。第四，果蝇中的Top3b突变体显示出异常的神经肌肉连接，与fMR1突变体中的连接类似。第五，果蝇中的TOP3B突变修饰了FMRP过表达引起的粗糙眼表型。第六和最重要的是，TOP3B可以直接催化RNA底物上的拓扑异构酶反应。另外，使其DNA拓扑异构酶活性失活的点突变也破坏了其RNA拓扑异构酶活性，表明可以将相同的催化残基用于DNA和RNA底物的反应。此外，TOP3B，TOP3A的旁系同源物完全缺乏RNA拓扑异构酶活性，这表明观察到的RNA拓扑异构酶活性是TOP3B的特异性。 最近，我们能够创建果蝇TOP3B/FMR1双重突变体，并发现在每个单个突变体中观察到的异常神经肌肉连接表型在双突变体中被抑制。这进一步说明了两种蛋白质以拮抗方式相互作用。此外，数据表明，RNA拓扑异构酶的抑制剂可用作减轻脆弱X患者疾病的药物。 最近的人类研究将TOP3B突变与精神分裂症，智力残疾和自闭症联系起来。与这些发现一致，我们发现TOP3B结合了由精神分裂症和自闭症相关基因编码的多个mRNA。我们进一步表明，一个与精神分裂症相关的基因PTK2/FAK显示出果蝇Top3b突变体，FMR1突变体及其双突变体的神经肌肉连接的表达降低，表明TOP3B和FMR1在同一途径中起作用，以促进PTK2表达在Synapse中的同一途径。我们还观察到果蝇和小鼠在TOP3B失活的果蝇和小鼠中的异常突触形成。 总而言之，我们已经将TOP3B确定为真核生物中不仅可以作用于DNA，还可以作用于RNA的第一个双活动拓扑酶。我们表明，TOP3B与FMRP合作，以促进神经发育和心理健康。描述这项工作的手稿已在自然神经科学中发表（Xu等人自然神经科学，2013年），并由自然，自然神经科学，自然评论神经病学以及其他期刊和组织中的亮点介绍。 一个重要的问题是，各种物种中的RNA拓扑异构酶活性如何流行。我们已经测试了各种物种的拓扑异构酶，发现RNA拓扑异构酶活性都存在于生命，细菌，古细菌和真核生物的所有三个领域。数据支持以下概念：RNA拓扑异构酶很重要，因此它们可以通过进化来保守。 我们和其他人先前已经表明，在人类伴侣的polybibosomes中，Top3b表明它参与了mRNA翻译。现在，我们发现来自其他动物物种的TOP3B也与多核糖体相关，而细菌和酵母中的Top3b则没有。此外，该关联需要TDRD3，这是TOP3B的合作伙伴，它仅存在于动物中。因此，我们的数据表明，仅在动物中，双重活性拓扑酶TOP3B作为复合物的一部分在mRNA翻译中起作用。 人细胞在细胞核和细胞质中有5种拓扑异构酶。我们发现只有TOP3B强烈结合mRNA。此外，我们发现这种结合活性在很大程度上取决于TOP3B的RNA结合域。这些数据与TOP3B充当RNA拓扑异构酶并在mRNA代谢中起作用的概念一致。 我们已经检查了在精神分裂症和自闭症患者中发现的TOP3B的两个从头单核苷酸变体，并发现自闭症患者衍​​生的点突变体失去了RNA拓扑异构酶的活性和mRNA结合活性。此外，两个突变体具有与FMRP相互作用的缺陷能力，数据为TOP3B参与精神疾病提供了更多证据。 我们产生了表达TOP3B不同突变体的转基因蝇。我们发现RNA结合活性和拓扑异构酶活性都是形成正常果蝇突触酶所必需的。此外，自闭症患者衍​​生的点突变体在促进突触形成方面有缺陷。数据支持以下概念：正常神经发育需要TOP3B的RNA拓扑蛋白酶酶活性。 我们已经表明，TOP3敲除小鼠表现出与精神疾病和认知障碍有关的行为表型，包括增加焦虑和恐惧，社交互动障碍以及缺陷的空间学习和记忆。另外，这些小鼠在成年海马神经发生和突触可塑性中显示出缺陷。值得注意的是，突变小鼠的大脑在响应恐惧调节压力时表现出受损的全球神经元活动依赖性转录，并且受影响的基因包括许多对于神经元功能和心理健康至关重要的。我们的数据表明，TOP3对于多个领域中的正常脑功能至关重要，并且有缺陷的神经元活动依赖性转录可能是TOP3突变引起认知障碍和精神疾病的机制。这项工作正在自然传播中进行修订。 我们发现，TDRD3不仅在人类，而且在果蝇中形成了TOP3B稳定的复合物。我们表明，在果蝇中，TOP3B生化和遗传与含有AGO2，P68 RNA Helicase和FMRP的RNAI诱导的沉默复合物（RISC）相互作用。 TOP3B和RISC突变体在异染色质形成和转录沉默中都有缺陷。在TOP3B和AGO2，P68和RNAi生物发生酶（DICER-2）之间的双重突变体中，该缺陷被抑制，表明TOP3B和siRNA机械之间的相互作用是协调的相互作用。此外，TOP3B和AGO2单突变蝇都表现出杂质和端粒异染色质中的异染色质标记降低。周围异染色质的降低在其双重突变体中也被抑制。此外，在TOP3B突变体中，端粒异染色质中几种基因的表达和转座元件增加。最后，我们发现TOP3B取决于其RNA结合结构域及其催化活性以促进异染色质形成和转录沉默。我们的数据表明，TOP3B可与siRNA机械一起促进异染色质形成和转录沉默。这项工作在自然界中。 我们已经在实验室建立了CRISPR-Cas9系统，并能够在果蝇中敲除TDRD3。我们发现TDRD3抑制了TOP3B功能，但与FMR1合作。 TDRD3苍蝇还表现出DA神经元的异常发育。他们的卵母细胞发育也有缺陷。我们现在正在研究基本机制。 我们已经建立了TDRD3突变小鼠系。在马克·马特森（Mark Mattson）实验室中与王·王（Yue Wang）合作，我们发现突变系列有缺陷的突触传播。我们还观察到杀伤力的高渗透率。