Novel inhibitor targets for ribosome biogenesis

Ribosomes are composed of a large (60S) and a small (40S) subunit and are the cellular machinery responsible for the translation of genetic information into proteins. The formation of ribosomes is a highly dynamic and energy consuming process and requires the participation of about 200 transacting factors that transiently associate with the pre-ribosomal particles at different maturation steps. Due to the dynamic nature of the process and the plethora of factors involved, our knowledge especially about very early maturation steps is limited. Since inhibitors proved to be valuable tools to study dynamic processes, we recently performed a high throughput screening to identify inhibitors of ribosome biogenesis. Indeed we could identify about 30 compounds affecting different steps of large ribosomal subunit formation in yeast. Aim of this project is to characterize the mode of action of selected inhibitors and to identify their target genes. For this project we will focus on those inhibitors blocking very early steps in large ribosomal subunit maturation since especially these early steps are not very well characterized up to now. The detailed characterization of the mode of action of the inhibitors and of the function of their targets will allow us to identify novel players in ribosome biogenesis and to expand our understanding of very early steps of the large subunit biogenesis pathway. In addition, we will investigate the impact of the inhibitors on the maturation cascade. The fast onset of inhibition of the pathway upon drug treatment will enable us to block the pre-ribosome assembly process very rapidly and hence to investigate the pathway with high temporal resolution, which is hardly possible with conventional methods. Therefore this project will also help to better understand the dynamics of the highly conserved ribosome assembly and maturation pathway. Since efficient ribosome assembly is especially important for fast growing cells, the targets identified by this project will also represent promising candidates for anti-tumor chemotherapy. Therefore the results from this project are likely to stimulate the development of novel ribosome biogenesis inhibitors for medical treatment of malignancies.

Ribosomes are responsible for the translation of the genetic information into the amino acid sequence of proteins. The formation of ribosomes is a major task in each eukaryotic cell and involves the activity of all three RNA polymerases. Due to its fundamental role, ribosome biogenesis is tightly coordinated with cell cycle control and energy metabolism. Ensuring a constant supply of freshly synthesized ribosomes is of special importance for fast proliferating cells which highlights ribosome biogenesis as promising pathway to target tumor cells. In addition, inhibitors of ribosome biogenesis represent valuable tools to investigate the highly dynamic formation of ribosomes step by step with high temporal resolution which cannot be achieved by other means. During this project we have characterized the mode of action of several selected novel inhibitors targeting the maturation of the large ribosomal subunit which we have identified in a microscopy-based screen (Awad et al., 2019). In order to understand the exact effect of these inhibitors, we have characterized the maturation state of isolated pre-ribosomes after inhibitor treatment of yeast cells by analyzing their protein and RNA composition. In addition we isolated and characterized mutants showing altered susceptibility to the drugs. Based on these analyses we found that the tested inhibitors block ribosome biogenesis at various stages providing us with a broad toolset to analyze the maturation pathway. For example, we could show that the lichen secondary metabolite Usnic acid inhibits the formation of large ribosomal subunits. This compound is of interest due to its pronounced anti-cancer activity but the mechanism behind this activity was unknown. The results of this project demonstrate that Usnic acid targets very early steps in ribosome formation, thereby explaining its anti-cancer activity. Our results thus show that ribosome biogenesis inhibitors not only provide valuable tools to better understand this highly dynamic pathway, but also have the potential to serve as anti-tumor drugs.