Investigation of an RNA topoisomerase complex involved in Fragile X syndrome

脆性 X 综合征中涉及的 RNA 拓扑异构酶复合物的研究

基本信息

  • 批准号:
    9147322
  • 负责人:
  • 金额:
    $ 64.4万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
  • 资助国家:
    美国
  • 起止时间:
  • 项目状态:
    未结题

项目摘要

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 RNA topoisomerase in eukaryotes and showed that it 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 are RNA topoisomerases in various species. We have tested topoisomerases from a variety of species, and found that RNA topoisomerases are 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. 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 collaborated with Yue Wang in Mark Mattson's lab, and found that Top3b mutant mice show abnormal synapse plasticity in both long-term potentiation and long-term depression. We also collaborated with Henrietta van Praag's lab and observed increased anxiety phenotype in Top3b knockout mice. Moreover, we worked with Richard Spencer's lab and found that Top3b mutant mice have increased ventricle size, which is a characteristic of schizophrenia patients. Furthermore, we found several early response genes have abnormal expression in the brains of Top3b knockout mice. Phosphorylation of several important signaling molecules is also abnormal in the brain of Top3b mutant mice. These data support the notion that Top3b is important for normal neurodevelopment, and its defect can lead to mental dysfunction. We found that TDRD3 forms a stable complex with Top3b in not only human, but also Drosophila. We have performed unbiased purification of the Top3b-TDRD3 complexs in both species, and found that they all associate with not only FMRP, but also other RNA processing enzymes. We have mapped the FMRP-interaction domain to a conserved motif in TDRD3, and are in the process to map other interaction domains. We have established CRISPR-CAS9 system in the lab, and were able to knockout TDRD3 in Drosophila. We found that the TDRD3 mutant flies display similar neuromuscular junction defect as the Top3b and Fmr1 mutants. Genetically, 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 used CRISPR-CAS9 system to inactivate TDRD3 in Drosophila S2 cells. We found that Top3b fails to interact with FMRP in these cells, indicating that TDRD3 serves as a bridge to connect Top3b and FMRP. We have established a TDRD3 mutant mouse line, and found that this line has no detectable expression of TDRD3, and reduced expression Top3b. In collaboration with Yue Wang in Mark Mattson's lab, we found that the TDRD3 mutant mice have abnormal synapse transmission. These data suggest that TDRD3 works with Top3b to promote neurodevelopment in both flies and mice.
拓扑异构酶是 DNA 世界的魔术师,利用其魔法解决 DNA 复制、修复和转录过程中的拓扑问题。许多 DNA 代谢酶(聚合酶、解旋酶、核酸酶和连接酶)在 RNA 领域都有对应物。拓扑异构酶是一个例外,RNA 世界中似乎不存在这种酶。 在我们对参与DNA修复的DNA拓扑异构酶的研究中,我们发现拓扑异构酶3β(Top3b)具有RNA拓扑异构酶的许多特征。首先,Top3b 与脆性 X 综合征蛋白 FMRP 相关,已知 FMRP 可以结合 mRNA 并调节 mRNA 翻译和运输。其次,Top3b 与 FMRP 相似,与多核糖体相关,多核糖体是 mRNA 翻译的单位。第三,Top3b 与 FMRP 共定位于 RNA 应激颗粒中,RNA 应激颗粒是停滞 mRNA 和翻译机制的细胞质区室。第四,交联 RNA 免疫沉淀测定 (HITS-CLIP) 显示 Top3b 与细胞中的 mRNA 结合。第四,果蝇中的 Top3b 突变体表现出与 FMR1 突变体相似的异常神经肌肉接头。第五,果蝇中的 Top3b 突变改变了 FMRP 过度表达诱导的粗糙眼表型。第六,也是最重要的一点,Top3b 可以直接催化 RNA 底物上的拓扑异构酶反应。此外,使其DNA拓扑异构酶活性失活的点突变也会破坏其RNA拓扑异构酶活性,表明相同的催化残基可能用于DNA和RNA底物上的反应。此外,Top3b 的旁系同源物 Top3a 完全缺乏 RNA 拓扑异构酶活性,表明观察到的 RNA 拓扑异构酶活性是 Top3b 特异的。 最近,我们能够创建果蝇Top3b/Fmr1双突变体,并发现在每个单突变体中观察到的异常神经肌肉接头表型在双突变体中受到抑制。这进一步说明这两种蛋白质在遗传上以拮抗方式相互作用。此外,数据表明RNA拓扑异构酶抑制剂可用作缓解脆性X染色体患者病情的药物。 最近的人类研究已将 Top3b 突变与精神分裂症、智力障碍和自闭症联系起来。与这些发现一致的是,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。我们进一步发现,一种精神分裂症相关基因 ptk2/FAK 在果蝇 Top3b 突变体、Fmr1 突变体及其双突变体的神经肌肉接头中表现出表达降低,表明 Top3b 和 Fmr1 在同一途径中发挥作用,促进突触中 ptk2 的表达。我们还在果蝇和小鼠中观察到 Top3b 失活的异常突触形成。 总之,我们已经确定 Top3b 是真核生物中第一个 RNA 拓扑异构酶,并表明它与 FMRP 一起促进神经发育和心理健康。描述这项工作的手稿已发表在《自然神经科学》(Xu et al. Nature Neuroscience,2013)上,并在《自然》、《自然神经科学》、《自然评论神经学》和其他期刊和组织中得到了重点报道。 一个重要的问题是 RNA 拓扑异构酶在不同物种中的流行程度。我们测试了多种物种的拓扑异构酶,发现 RNA 拓扑异构酶存在于生命的所有三个领域:细菌、古细菌和真核生物中。这些数据支持这样的观点:RNA 拓扑异构酶很重要,因此它们在进化过程中得以保守。 人体细胞的细胞核和细胞质中有5种拓扑异构酶。我们发现只有 Top3b 与 mRNA 强烈结合。此外,我们发现这种结合活性强烈依赖于 Top3b 的 RNA 结合结构域。这些数据与 Top3b 作为 RNA 拓扑异构酶并参与 mRNA 代谢的观点一致。 我们检查了在精神分裂症和自闭症患者中发现的 Top3b 的两种从头单核苷酸变异,发现源自自闭症患者的点突变体失去了 RNA 拓扑异构酶活性和 mRNA 结合活性。此外,两种突变体与 FMRP 相互作用的能力都有缺陷。这些数据为 Top3b 参与精神障碍提供了额外的证据。 我们生产了表达 Top3b 不同突变体的转基因果蝇。我们发现RNA结合活性和拓扑异构酶活性都是正常果蝇突触酶形成所必需的。此外,自闭症患者衍​​生的点突变体在促进突触形成方面存在缺陷。这些数据支持以下观点:正常神经发育需要 Top3b 的 RNA 拓扑异构酶活性。 我们与Mark Mattson实验室的Yue Wang合作,发现Top3b突变小鼠在长时程增强和长时程抑制方面都表现出异常的突触可塑性。我们还与 Henrietta van Praag 的实验室合作,观察到 Top3b 基因敲除小鼠的焦虑表型增加。此外,我们与Richard Spencer的实验室合作发现,Top3b突变小鼠的心室尺寸增大,这是精神分裂症患者的一个特征。此外,我们发现一些早期反应基因在Top3b基因敲除小鼠的大脑中表达异常。 Top3b 突变小鼠大脑中几种重要信号分子的磷酸化也出现异常。这些数据支持这样的观点:Top3b 对于正常神经发育很重要,其缺陷可能导致精神功能障碍。 我们发现 TDRD3 不仅在人类中,而且在果蝇中与 Top3b 形成稳定的复合物。我们对两个物种的 Top3b-TDRD3 复合物进行了无偏纯化,发现它们不仅与 FMRP 相关,还与其他 RNA 加工酶相关。我们已经将 FMRP 相互作用域映射到 TDRD3 中的保守基序,并且正在映射其他相互作用域。 我们在实验室建立了CRISPR-CAS9系统,并能够在果蝇中敲除TDRD3。我们发现 TDRD3 突变体果蝇表现出与 Top3b 和 Fmr1 突变体相似的神经肌肉接头缺陷。从基因上来说,TDRD3 抑制 Top3b 功能,但与 Fmr1 协同工作。 TDRD3 果蝇还表现出 DA 神经元发育异常。他们的卵母细胞发育也有缺陷。我们现在正在研究其根本机制。 我们使用 CRISPR-CAS9 系统灭活果蝇 S2 细胞中的 TDRD3。我们发现Top3b在这些细胞中无法与FMRP相互作用,表明TDRD3充当了连接Top3b和FMRP的桥梁。 我们建立了TDRD3突变小鼠品系,发现该品系没有检测到TDRD3的表达,并且Top3b的表达减少。我们与 Mark Mattson 实验室的 Yue Wang 合作,发现 TDRD3 突变小鼠具有异常的突触传递。这些数据表明 TDRD3 与 Top3b 一起促进果蝇和小鼠的神经发育。

项目成果

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Weidong Wang其他文献

Weidong Wang的其他文献

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{{ truncateString('Weidong Wang', 18)}}的其他基金

Developing proinsulin misfolding inhibitors for beta cell protection and diabetes treatment
开发用于 β 细胞保护和糖尿病治疗的胰岛素原错误折叠抑制剂
  • 批准号:
    10529960
  • 财政年份:
    2022
  • 资助金额:
    $ 64.4万
  • 项目类别:
Developing proinsulin misfolding inhibitors for beta cell protection and diabetes treatment
开发用于 β 细胞保护和糖尿病治疗的胰岛素原错误折叠抑制剂
  • 批准号:
    10665748
  • 财政年份:
    2022
  • 资助金额:
    $ 64.4万
  • 项目类别:
Preclinical Validation of PPARg Acetylation Inhibitors for Diabetes Prevention and Treatment
PPARg 乙酰化抑制剂预防和治疗糖尿病的临床前验证
  • 批准号:
    10580851
  • 财政年份:
    2021
  • 资助金额:
    $ 64.4万
  • 项目类别:
Cytoprotection and the mechanism of action of a natural product Khellin against ER stress
天然产物 Khellin 对抗 ER 应激的细胞保护和作用机制
  • 批准号:
    9974514
  • 财政年份:
    2018
  • 资助金额:
    $ 64.4万
  • 项目类别:
Cytoprotection and the mechanism of action of a natural product Khellin against ER stress
天然产物 Khellin 对抗 ER 应激的细胞保护和作用机制
  • 批准号:
    10285537
  • 财政年份:
    2018
  • 资助金额:
    $ 64.4万
  • 项目类别:
Preclinical Development of Khellin Analogs for Anti-Diabetic Therapy
用于抗糖尿病治疗的 Khellin 类似物的临床前开发
  • 批准号:
    9336063
  • 财政年份:
    2016
  • 资助金额:
    $ 64.4万
  • 项目类别:
Preclinical Development of Khellin Analogs for Anti-Diabetic Therapy
用于抗糖尿病治疗的 Khellin 类似物的临床前开发
  • 批准号:
    9353780
  • 财政年份:
    2016
  • 资助金额:
    $ 64.4万
  • 项目类别:
Structural And Functional Studies Of Human Swi/snf Chromatin-remodeling
人类 Swi/snf 染色质重塑的结构和功能研究
  • 批准号:
    10467892
  • 财政年份:
  • 资助金额:
    $ 64.4万
  • 项目类别:
Investigating the roles of Topoisomerase 3b-TDRD3 complex in neurodegeneration and Alzheimer's disease
研究拓扑异构酶 3b-TDRD3 复合物在神经退行性疾病和阿尔茨海默病中的作用
  • 批准号:
    10469229
  • 财政年份:
  • 资助金额:
    $ 64.4万
  • 项目类别:
Identification and Characterization of human Rsc Chromatin-Remodeling Complex
人类 Rsc 染色质重塑复合物的鉴定和表征
  • 批准号:
    6431445
  • 财政年份:
  • 资助金额:
    $ 64.4万
  • 项目类别:

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