RNA interference of Trypanosoma brucei topoisomerase IB: Both subunits are essential

Type IB topoisomerases are enzymes essential for the orderly synthesis of nucleic acids and are the molecular target for antitumor camptothecins. In dozens of organisms, including eukaryotes, bacteria, and viruses, this enzyme is monomeric. However, we previously found that topoisomerase IB in trypanosomes is a heteromultimer, comprised of two distinct subunits encoded by separate genes. A large 90 kDa subunit contains the DNA binding domain and a small 36 kDa subunit contains the catalytic domain. In this study we use RNA interference to silence each of the subunits separately. For each subunit, tetracycline-induced expression of double-stranded RNA results in drastic reduction of cognate mRNA and protein. For the large subunit, nucleic acid biosynthesis (as monitored by the incorporation of radiolabeled precursors into DNA and RNA) is halved by 39 h, and cell growth halts by 72 h, after induction. The steady state level of both nuclear and mitochondrial mRNAs is reduced. Virtually identical results are obtained by silencing the small subunit. Interestingly, although interference is specific at the level of mRNA, silencing of one subunit leads to a profound reduction in the level of protein for both subunits, suggesting that survival, or perhaps synthesis, of each subunit depends upon the presence of the other. These findings underscore the essential nature of type IB topoisomerase activity in Trypanosoma brucei and its suitability as a target for rational drug design.