Figure 4. Top3β has RNA topoisomerase activity that depends on a conserved RGG RNA-binding motif.

(a) Schematic representation of an RNA topoisomerase assay modified from a previous publication². A synthetic circular RNA substrate contains two pairs of complementary regions (red and green) separated by single-stranded spacers (black). Through strand passage reactions, this substrate is converted to a knot in which the two pairs of complementary regions can form normal double helices. (b) Silver-stained SDS gel showing purified recombinant wildtype or Y336F mutant HF-Top3β proteins. This mutation is known to inactivate topoisomerase activity on DNA. (c) Autoradiographs from the RNA topoisomerase assay show that Top3β, but not its catalytic mutant or Top3α, has RNA topoisomerase activity. The reaction contains 1 nM ³²P-labeled circular RNA substrate and increasing concentrations of wildtype or Y336F Top3β mutant (0.05 nM, 0.1 nM, 0.2 nM, 0.4 nM, 0.8 nM or 1.6 nM), or Top3α (0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM or 30 nM). A ladder of RNA markers and the purified RNA knot were loaded on the left of every panel. The linear breakdown products of cyclic RNA exist in all reactions. A darker exposure of the autoradiographs shows that a small amount of catenane was also generated in the reaction (Supplementary Fig. 4a). (d) Top panel: schematic representations showing that Top3β, but not Top3α, contains a RGG motif. Bottom panel: the alignment of RGG motifs of Top3β from several higher eukaryotes. The RGG boxes are indicated by underline. Arginine and Glycine are indicated by red and green letters, respectively. Conserved regions are highlighted in yellow. (e,f) Gel-shift assay (e) and its quantification (f) show that a fusion protein containing MBP and the RGG motif of Top3β (MBP-RGG) preferentially binds RNA compared to DNA. Reaction contains 1 nM single-stranded (ss) or double-stranded (ds) RNA or DNA. MBP-TDRD3_1-187 was included as a negative control. (g,h) RNA topoisomerase assay (g, right panel) and its quantification (h) show that the RGG motif of Top3β is important for its RNA topoisomerase activity; (g, left panel), silver-stained SDS gel of the purified wild-type or RGG motif-deleted Top3β. The representative images shown have been repeated at least twice, and the results are reproducible.