Selective inhibition of RNA polymerase I transcription as a potential approach to treat African trypanosomiasis

Abstract

Trypanosoma brucei relies on an essential Variant Surface Glycoprotein (VSG) coat for survival in the mammalian bloodstream. High VSG expression within an expression site body (ESB) is mediated by RNA polymerase I (Pol I), which in other eukaryotes exclusively transcribes ribosomal RNA genes (rDNA). As T. brucei is reliant on Pol I for VSG transcription, we investigated Pol I transcription inhibitors for selective anti-trypanosomal activity. The Pol I inhibitors quarfloxin (CX-3543), CX-5461, and BMH-21 are currently under investigation for treating cancer, as rapidly dividing cancer cells are particularly dependent on high levels of Pol I transcription compared with nontransformed cells. In T. brucei all three Pol I inhibitors have IC50 concentrations for cell proliferation in the nanomolar range: quarfloxin (155 nM), CX-5461 (279 nM) or BMH-21 (134 nM) compared with IC50 concentrations in the MCF10A human breast epithelial cell line (4.44 μM, 6.89 μM or 460 nM, respectively). T. brucei was therefore 29-fold more sensitive to quarfloxin, 25-fold more sensitive to CX-5461 and 3.4-fold more sensitive to BMH-21. Cell death in T. brucei was due to rapid inhibition of Pol I transcription, as within 15 minutes treatment with the inhibitors rRNA precursor transcript was reduced 97-98% and VSG precursor transcript 91-94%. Incubation with Pol I transcription inhibitors also resulted in disintegration of the ESB as well as the nucleolus subnuclear structures, within one hour. Rapid ESB loss following the block in Pol I transcription argues that the ESB is a Pol I transcription nucleated structure, similar to the nucleolus. In addition to providing insight into Pol I transcription and ES control, Pol I transcription inhibitors potentially also provide new approaches to treat trypanosomiasis.

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

“…For example, the increased VSG mRNA half-life (4.5 hrs) compared with ESAG 6 and 7 (1.8-2.8 hrs) [83,84]. Transcripts of genes located PNUTS-PP1 regulates transcription termination in T. brucei approximately 5 kb upstream of the VSG have also been shown to be selectively rapidly degraded [85]. We also noticed that increased levels of RNA close to the promoter are significantly higher when there is an additional BES promoter upstream for ESAG10.…”

Transcription-coupled repair inDrosophila melanogasteris independent of the mismatch repair pathway

“…TCR detects the base damage from RNA polymerase stalling in actively transcribed DNA (4, 5) and leads to a mutational asymmetry between the strands (6). Transcription inhibition can be dangerous to a cell or even an organism (7, 8) so a quick resumption of transcription is vital. TCR is found in most bacterial species and many eukaryotes (9) and a defective pathway causes strong disease phenotypes, such as xeroderma pigmentosum and Cockayne’s syndrome in humans (10).…”

Conserved nucleolar stress at the onset of cell death

“…Early rRNA synthesis inhibition and nucleolar disorganization were relatively easy to evidence in Dictyostelium cell death. Similar death‐associated steps seemed to occur also in other protists, since Pol I inhibitors caused Trypanosoma brucei cell death, together with within 15 min almost complete reduction in rRNA transcription and within 1 h nucleolus fragmentation . Chaetocin, a histone methyltransferase inhibitor, decreased Trypanosoma cruzi viability and promoted in particular nucleolus fragmentation associated with impairment of RNA transcription .…”