A dynamic view on ribosome formation

Ribosomes are the cellular machineries that produce proteins. The process of synthesizing new ribosomes, called ribosome biogenesis, is an essential and highly energy consuming task for all living cells. In eukaryotic cells more than 200 additional protein factors are needed to assemble ribosomal proteins with ribosomal RNAs (rRNAs) to form a functioning ribosome. This whole process starts in the nucleolus, a sub-compartment of the nucleus and is finalized in the cytoplasm. All along the way, the protein and RNA components are remodeled and refined and the rRNA elements are enzymatically processed in numerous consecutive steps. Since ribosome biogenesis has to be coordinated with cell growth and proliferation it underlies a tight but dynamic regulation. Only if the complete network of the 200 maturation factors acts in a timely and spatially coordinated manner, functional ribosomes can be formed. Any disturbance of the pathway affects the dynamics of the whole process. In the last decades, this pathway was approached by a combination of genetic, biochemical and structural biology methods, which resulted in amazing insights in individual stages of the ribosomal maturation pathway. However, we are still lacking sophisticated investigations that are able to capture the dynamics of this process in order to fully understand its sequence of events and regulation. To close this gap we have designed a strategy to target the ribosome biogenesis pathway by punctual inhibition with small molecular weight inhibitors. This inhibition acting at a scale of seconds allows us to dissect and quantify the individual maturation steps with unprecedented timely resolution. We will be able to address the association and dissociation of individual factors with pre-ribosomal particles, define functional linkages between these factors and uncover the functions of hitherto undefined maturation factors. Our goal is to draw a conclusive picture that displays the dynamic and interwoven network of all factors that contribute to the formation of eukaryotic ribosomes. Since ribosome biogenesis is an essential prerequisite for fast proliferation it is highly upregulated in cancer cells. Several commonly used chemotherapeutic agents directly or indirectly affect the regulation of ribosome formation. Based on this knowledge, a detailed understanding of this regulation can help to design novel strategies for anti-cancer chemotherapy.

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 which is tightly coordinated with cell cycle control and energy metabolism. The process is of special importance for fast dividing cells which makes ribosome biogenesis to a promising target for inhibition of fast dividing cells, like 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. During this project we aimed to better understand and characterize the individual steps of ribosome biogenesis and how they are interconnected. To achieve this goal, we made use of several novel ribosome biogenesis inhibitors that we identified previously. This enabled us to extremely rapidly inhibit the assembly process. This strategy allowed us for the first time to dissect primary effects of the inhibition of particular steps of ribosome biogenesis from secondary and tertiary consequences. Our investigations provided us with detailed insights into the exact temporal order of the individual assembly reactions, their dynamics and how the individual steps are interconnected. These investigations were expanded by determining the cellular concentration and distribution of key assembly factors, which for the first time enabled a quantitative understanding of the individual ribosome assembly steps.