The ribosome assembly path of Rps3

Ribosomes are the universal protein synthesis machines consisting of ribosomal RNA (rRNA) and ribosomal proteins. The synthesis of ribosomes out of these building blocks is a central process in every growing cell. The assembly of ribosomal proteins with rRNA occurs in the nucleus; however, ribosomal proteins are synthesized in the cytoplasm. The fast rates of ribosome biogenesis necessitate mechanisms to promote their efficient nuclear import, prevent non-specific interactions, and ensure the correct assembly with the rRNA. In this project, we will study the assembly path of ribosomal proteins using Rps3, a protein of the small ribosomal subunit as model. We found that newly synthesized Rps3 is bound by its specific chaperone Yar1. Furthermore, we made the intriguing observation that Rps3 is dimeric in this complex. Rps3 is imported into the nucleus by importin a/ß, and assembled to pre-ribosomal particles in complex with Yar1. Lateron, Rps3 interacts with the assembly factor Ltv1. All these interaction partners have overlapping or adjacent binding sites within the N-terminal a-helix of Rps3. In the first part of the project, we will investigate the functions of these interactions in more detail and determine when and how the individual factors bind and leave Rps3 during the maturation path. To understand the modes of interaction and how Rps3 is handed over from one partner to the next, we will obtain crystal structures of the complexes of Rps3 bound to importin a and Rps3 bound to Ltv1 and draw a structural comparison of the interactions of Rps3 with different partners. The third part of this project will be dedicated to the further investigation of the dimeric conformation of Rps3. We will address how these dimers are formed and released. Complex conformations and interaction networks like those observed for Rps3 may also play a role in other cellular assembly processes. Therefore the structural and functional dissection of the assembly process of Rps3 could also inspire the investigation of assembly processes of other ribosomal and non-ribosomal proteins. As the composition of ribosomes and the ribosome biogenesis machinery are highly conserved between yeast and humans, many of the obtained results will not only apply to yeast, but also to humans. Recent data suggest that ribosome biogenesis and in particular, the assembly of ribosomal proteins, has a critical role in anaemia and cancer. Therefore, the understanding of the fundamental process of ribosome biogenesis will on a long term be important for the molecular understanding and treatment of human diseases.

All proteins in a cell are manufactured by a highly efficient and accurate nano-machine, the ribosome. Each cell contains more than 100,000 ribosomes, which are continously engaged in protein production. This also means that whenever a cell duplicates, more than 100,000 new ribosomes have to be synthesized out of their building blocks, the ribosomal RNAs (rRNAs) and ribosomal proteins (r-proteins). These ribosomal constituents are produced in two different cellular compartments. While rRNA is synthesized in the nucleus, r-proteins are produced in the cytoplasm and have to be transported into the nucleus, where they bind to the rRNA. In this project, we exemplarily characterized the path of one r-protein, Rps3, from its synthesis to its stable incorporation into the ribosome. Surprisingly, we discovered that newly synthesized Rps3 forms a dimer (two Rps3s binding to each other). We speculate that this conformation makes Rps3 more stable during its transport. The free ends of this Rps3 dimer are bound by two different proteins, a "bodyguard" protecting Rps3 from undesired encounters with other molecules, and a vehicle (called importin) that transports the whole complex into the nucleus. There, Rps3 encounters rRNA and the bodyguard is finally removed upon displacement by another protein. We also found that two other r-proteins, Rps6 and Rps2, have their own bodyguards. Moreover, Rps2 seems to utilize partially similar mechanisms as Rps3 in order to reach the nucleus and become incorporated into the emerging ribosomes. Our study highlights that a growing cell is not only challenged by the task of synthesizing large amounts of r-proteins, but in addition to that, it also needs to ensure that they are savely guided to the place where they come into contact with the rRNA. Only if these individual steps have been executed correctly, a functional ribosome is generated and can join its fellow ribosomes to maintain the required rates of protein synthesis in a cell.