Assembly factor release in 40S ribosomal subunit maturation

Ribosomes are extremely important nanomachines responsible for protein synthesis in every cell. The demand for new proteins is so high that each cell needs at least 100,000 ribosomes, most of them being constantly active in protein production. Imporantly, also the ribosomes themselves have to be synthesized in the cell, especially when a cell divides and has to provide ~100,000 new ribosomes to the offspring. Therefore, ribosome synthesis is a central process in every growing cell, and mistakes in this process are connected to diseases like cancer and bone marrow failure syndroms. For these reasons, it is very important to understand how this pathway works. Each ribosome is composed of the same building blocks: four ribosomal RNAs and about 80 different ribosomal proteins. The synthesis of ribosomes can be imagined as a mechanical construction work in which many different workers put together the individual components in the correct order and shape the structure of the ribosome. There are more than 200 different workers, termed "ribosome assembly factors", engaged in the production of each single ribosome. Most of these assembly factors are tightly bound to the emerging ribosome while they are doing their work, and the cell has to ensure that they are removed again once their work has been done. This is very important as the presence of these assembly factors can prevent further work by other assembly factors, but can also prevent ready-made ribosomes from functioning correctly. In this project, we want to decipher how the cell coordinates the stepwise shaping of ribosomes with the removal of assembly factors. To find out more about assembly factor removal, we will generate yeast mutants in which the removal of individual assembly factors is blocked. Like that, we can freeze the synthesis process at the step where this factor is normally removed. By analysing what is going wrong in these mutants, we can then improve our understanding of how this process works. Moreover, by structural analysis we can investigate how these "frozen" ribosome precursors look like. By performing such analyses with several different mutants blocked in different steps of the process, we can then analyse how the composition and structure of ribosome precursors changes. By connecting the information from all these different "frozen" intermediates, we want to finally assemble a kind of "slow motion video" of the ribosome synthesis process. The results of this work are expected to lead to a better understanding of how the process of ribosome synthesis works and will consequently also help to find out what is going wrong in diseases caused by defective ribosome synthesis.

Ribosomes are tiny, essential machines found in every living cell, responsible for producing the proteins that our bodies need to function properly. Because these machines are so crucial, cells must assemble them with great care through a complex process known as ribosome biogenesis. Errors in this process can have serious consequences, leading to severe health issues such as cancer and bone marrow disorders. To gain a deeper understanding of how ribosomes are built, we are investigating this process in yeast, which shares key similarities with humans in how ribosomes are made. Each ribosome is made up of four ribosomal RNAs and approximately 80 different ribosomal proteins. These building blocks must be put together in a precise order, guided by over 200 specialized helper proteins known as ribosome assembly factors. Given that these assembly factors operate in different regions of the ribosome, we sought to understand how their actions are coordinated and how ribosomal proteins contribute to this intricate process. To delve into this, we focused on the interactions between assembly factors and ribosomes during the final stages of assembly. Our research uncovered that a ribosomal protein called Rps15 plays a pivotal role in these late stages, interacting with several assembly factors. Mutations in Rps15 disrupt these crucial steps, leading to the production of malfunctioning ribosomes with a higher error rate in protein synthesis. This is particularly relevant because similar mutations have been found in patients with chronic lymphatic leukemia. Our discovery underscores the importance of studying Rps15, not only to deepen our understanding of ribosome assembly, but also to unravel the underlying causes of chronic lymphatic leukemia, to be able to come up with better treatment options in the future. Additionally, our work explored how ribosome assembly factors eventually detach from the ribosome, a key step that allows the ribosome to become fully functional. We discovered that this process requires a series of carefully coordinated interdependent events, involving intricate communication between assembly factors. If any part of this maturation cascade is disrupted, the entire process is halted, preventing the final release of the assembly factors. Only after this release can one of the last critical building blocks, the ribosomal protein Rps10, be successfully integrated into the ribosome. In summary, our research has provided significant new insights into the complex process of ribosome assembly. These finding have the potential to enhance our understanding of diseases like chronic lymphatic leukemia and could pave the way for new approaches to preventing and treating such conditions in the future.