The function of the ribosomal protein S15 in 40S subunit biogenesis

Ribosome biogenesis is the process responsible for correctly assembling ribosomal RNAs and ribosomal proteins into functional, translation competent ribosomes. This is one of the most energy consuming processes in a cell that takes place in the nucleolus, the nucleoplasm and the cytoplasm. In addition to their structural function in mature ribosomes, ribosomal proteins also play important roles in ribosome biogenesis. Mutations in various human ribosomal proteins have been reported to result in diseases like bone marrow failure syndromes, making ribosomal proteins and their involvement in ribosome biogenesis an interesting subject to study. Up to now, for only few ribosomal proteins detailed functions in ribosome biogenesis have been described. The aim of the proposed project is to investigate the function of Rps15, a ribosomal protein of the small (40S) ribosomal subunit, in ribosome biogenesis. The studies will be performed in the yeast Saccharomyces cerevisiae, which is due to the combination of powerful genetic and biochemical methods the most commonly used and best studied model organism for eukaryotic ribosome biogenesis. In preliminary experiments, we identified Rps15 as a genetic interaction partner of a known 40S maturation factor, suggesting it could also be involved in ribosome biogenesis. Our current hypothesis based on preliminary data as well as the recently published crystal structure of a eukaryotic 40S subunit predicts that Rps15 is involved in a structural re-arrangement that takes place in a late stage of 40S biogenesis. The characterization of Rps15 in the course of this project will include the generation of various mutants, and their phenotypic investigation in respect to defects in late 40S biogenesis. Especially, we will put efforts in identifying and characterizing structural differences of 40S subunits in the mutants as compared to wild-type cells. The proposed experiments will allow us to gain an understanding of the role of Rps15 in the ribosome biogenesis process. Furthermore, the characterization of the role of Rps15 in structural re-arrangements during the 40S biogenesis pathway would be an important step to a better understanding of the structural changes ribosomal particles undergo in course of their maturation. Further, as Rps15 is highly conserved from yeast to human, our results will also help understand the function of the human orthologue, and might in the end contribute to a better understanding of the link between ribosome biogenesis defects and diseases.

Ribosomes are large and complex factories responsible for the production of all proteins in a cell. They are composed of a large and a small subunit, which are each built up of ribosomal RNA and ribosomal proteins. The subject of this project was to investigate how ribosomes are synthesized. Ribosome synthesis is a highly important process in every growing cell, as the whole ribosome population of a cell (at least 100,000 ribosomes) has to be duplicated with every cell division. Ribosome synthesis starts with the assembly of ribosomal RNA and ribosomal proteins into ribosomal precursor particles. These initial precursors undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. It was previously observed that some ribosomal proteins, including the small subunit protein Rps3, are initially only weakly bound to ribosome precursor particles, and that they become tighter associated in the course of ribosome maturation. The reason for this phenomenon was however up to now unclear. Rps3 is composed of two globular domains, separated by a short loop. We propose that initially, only one of these domains (the C-domain of Rps3) is incorporated into small subunit precursors, while the other domain (the N-domain) protrudes from the surface of the precursor particle. Moreover, our data suggest that the ribosome assembly factor Ltv1 fixes the N-domain of Rps3 in this orientation while shielding its interaction sites within the ribosomal RNA and ribosomal protein Rps20. Consequently, Ltv1 release from small subunit precursor particles is necessary to allow for the full incorporation of Rps3. Ltv1 release is accomplished by a two step mechanism. Phosphorylation of Ltv1 presumably leads to an electrostatic repulsion of the protein from its binding site. Then, flipping of the Rps3 N-domain into its final position and formation of interactions of Rps3 with ribosomal RNA and ribosomal protein Rps20 leads to the eventual release of Ltv1 and results in the stable integration of Rps3. This stepwise assembly of two domains povides an explanation how the interaction of Rps3 with small subunit precursor particles is tightened in the course of ribosome synthesis. Stepwise assembly of individual domains may represent a new paradigm for the incorporation of ribosomal proteins.