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. 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.