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 a topoisomerase that has many features of an RNA topoisomerase. First, this topoisomerase associates with the Fragile X syndrome protein, FMRP, which is known to bind mRNA and to regulate mRNA translation and transport. Second, the topiosomerase resembles FMRP in associating with polyribosomes, which are units for mRNA translation. Third, the topoisomerase colocalizes with FMRP in RNA stress granules, which are cytoplasmic compartments for stalled mRNA and translation machinery. Fourth, the topoisomerase binds mRNA in cells as shown by a crosslinked-RNA immunoprecipitation assay (HITS-CLIP). Fourth, the topoisomerase mutants in Drosophila display abnormal neuromuscular junctions similar to those in FMR1 mutants. Fifth, the topoisomerase mutations in Drosophila modify the rough eye phenotype induced by FMRP over-expression. Sixth and most importantly, this topoisomerase can directly catalyze topoisomerase reactions on RNA substrates. In addition, a point mutation that inactivates its DNA topoisomerase activity also lacks the RNA topoisomerase activity, indicating that the same catalytic residue may be used for both DNA and RNA substrates. Furthermore, a different human DNA topoisomerase completely lacks RNA topoisomerase activity, suggesting that the observed RNA topoisomerase activity is specific for our protein. During the past year, we were able to create double mutant of Drosophila that lacks both the RNA topoisomerase and FMRP. 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. Moreover, the data suggest that the inhibitors of the RNA topoisomerase may be used as drugs to alleviate conditions of the Fragile X patients. In addition, we were able to show that the abnormal neuromuscular junction phenotype can be rescued by wildtype topoisomerase, but not by a catalytic mutant, indicating that the topoisomerase activity per se is required for neuronal development in vivo. In summary, we have identified the first RNA topoisomerase in eukaryotes and showed that it antagonizes function of FMRP. A manuscript describing this work has been submitted for publication. We are now using a combination of biochemistry and genetics approaches to identify the target mRNAs for this topoisomerase. The goal is to identify the specific target mRNAs that are relevant to longevity and Fragile X syndrome.
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