Investigation of an RNA topoisomerase complex involved in Fragile X syndrome

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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.