Topoisomerases resolve topological problems generated during DNA metabolism (Wang et al. NRMCB 2002), but the roles of topoisomerases in RNA metabolism remain unclear. Our prior study has identified human Topoisomerase 3b (Top3b) as the first RNA topoisomerase in eukaryotes (Xu et al. 2013), whereas another study has linked Top3b gene deletion to schizophrenia and intellectual disability (Stoll et al. 2013). Mechanistically, Top3b forms a stoichiometric complex with TDRD3 (Tudor domain containing 3); and a fraction of this complex associates with FMRP (Xu et al. 2013), an RNA-binding protein known to regulate translation of mRNAs important for synapse development and autism. FMRP is encoded by the fmr1 gene, which is inappropriately silenced in the Fragile X mental retardation syndrome, a leading cause of autism. Using Drosophila as a model, we showed that Top3b genetically interacts with fmr1 to promote synapse formation.
Increasing evidence has shown that Drosophila FMRP is a component of the RNAi-induced silencing complex (RISC), which includes AGO2, p68 helicase, and Vig. Mutations in fmr1, other components of RISC, as well as Dicer-2 (Dcr-2; a protein essential for siRNA biogenesis), disrupt heterochromatin formation, transcriptional gene silencing, and repression of transposable elements. Here, we show that similar to FMRP, the Drosophila Top3b-TDRD3 complex also stably associates with RISC; and mutation of Top3b disrupts heterochromatin formation and transcriptional silencing in Position Effect Variegation (PEV) reporter assays. In addition, Top3b genetically interacts with AGO2, p68, and Dcr-2 in PEV assays, indicating that Top3b works coordinately with the siRNA machinery to facilitate heterochromatic gene silencing. Moreover, an epigenetic marker of heterochromatin, H3K9me2, displayed abnormal distribution in Top3b mutant flies. Furthermore, microarray and RT-qPCR analyses revealed that Top3b mutant flies exhibit global derepression of many genes in the pericentric heterochromatin region, as well as in other regions of different chromatin states. Finally, several transposable elements are de-silenced in the Top3b mutant flies. Together, our data suggest that Top3b interacts with the siRNA machinery to promote heterochromatin formation and transcriptional silencing.