Investigation of an RNA topoisomerase complex involved in Fragile X syndrome

拓扑异构酶是DNA世界的魔术师，在复制、修复和转录过程中解决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世界中都有对应物。一个例外是拓扑异构酶，它似乎在RNA世界中不存在。 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3 β（Top 3b）具有RNA拓扑异构酶的许多特征。首先，Top 3b与脆性X综合征蛋白FMRP相关，已知FMRP结合mRNA并调节mRNA翻译和转运。第二，Top 3b类似于FMRP与多聚核糖体，这是mRNA翻译的单位。第三，Top 3b与FMRP共定位在RNA应激颗粒中，RNA应激颗粒是停滞的mRNA和翻译机器的细胞质隔室。第四，Top 3b结合细胞中的mRNA，如通过交联RNA免疫沉淀测定（HITS-CLIP）所示。第四，果蝇Top 3b突变体显示异常的神经肌肉接头类似于FMR 1突变体。第五，果蝇中的Top 3b突变修饰了由FMRP过表达诱导的粗糙眼表型。第六，也是最重要的，Top 3b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破坏其RNA拓扑异构酶活性，表明相同的催化残基可用于DNA和RNA底物上的反应。此外，Top 3b的部分，Top 3a，完全缺乏RNA拓扑异构酶活性，表明所观察到的RNA拓扑异构酶活性是特异性的Top 3b。 在过去的一年中，我们能够创建果蝇Top 3b/Fmr 1双突变体。有趣的是，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被抑制。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拓扑异构酶的抑制剂可以用作药物来缓解脆性X患者的状况。 最近的人类研究将Top 3b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Top 3b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，一个精神分裂症相关的基因，ptk 2/FAK，显示在果蝇Top 3b突变体，Fmr 1突变体，和他们的双突变体的神经肌肉接头的表达减少，这表明Top 3b和Fmr 1工作在相同的途径，以促进ptk 2在突触中的表达。我们还观察到果蝇和小鼠中Top 3b失活的异常突触形成。 总之，我们已经确定Top 3b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》上，并在《自然》、《自然神经科学》、《自然评论神经病学》和其他期刊和组织上重点介绍。 我们现在正在使用生物化学和遗传学方法的组合来阐明Top 3b和FMRP如何共同调节基因表达。 一个重要的问题是RNA拓扑异构酶在不同物种中的普遍程度。我们已经测试了来自各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。这支持了RNA拓扑异构酶是重要的，因此它们在进化过程中是保守的。 我们已经检查了在精神分裂症和自闭症患者中发现的Top 3b的两种从头单核苷酸变体，发现它们要么失去了RNA拓扑异构酶活性，要么失去了与FMRP相互作用的能力，或者两者兼而有之。这些数据为Top 3b参与精神障碍提供了额外的证据。 我们产生了表达Top 3b的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。这些数据支持Top 3b的RNA拓扑异构酶活性是正常神经发育所必需的这一观点。

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. During the past year, we were able to create Drosophila Top3b/Fmr1 double mutant. Interestingly, 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, and is featured by highlights in Nature, Nature Neuroscience, Nature Review Neurology, and other journals and organizations. We are now using a combination of biochemical and genetic approaches to elucidate how Top3b and FMRP work together to regulate gene expression. 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 support the notion that the RNA topoisomerases are important so that they are conserved through evolution. We have examined two de novo single nucleotide variants of Top3b discovered in schizophrenia and autism patients, and found that they either lost the RNA topoisomerase activity, or the ability to interact with FMRP, or both. 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. The data support the notion that the RNA topoiomerase activity of Top3b is needed for normal neurodevelopment.