Paving the way to the rRNA - assembly factors for ribosomal proteins

Ribosomes are universal protein synthesis machines composed of a large and a small subunit. The highly complex structure of ribosomes is sustained by ribosomal proteins, which interact with the negatively charged ribosomal RNA (rRNA). Ribosomal proteins are synthesized in the cytoplasm, however incorporation of most ribosomal proteins into ribosomal subunits takes place in the nucleus. This spatial separation together with the high synthesis rates of ribosomes necessitate mechanisms to ensure that ribosomal proteins are efficiently delivered to their assembly site in the nucleus. Our preliminary data show that the yeast ribosomal protein Rps3 forms a stable complex with the non-ribosomal protein Yar1 before its incorporation into ribosomes. This interaction is important for the solubility and for efficient ribosome assembly of Rps3. By affinity purification, we identified additional non-ribosomal interaction partners of ribosomal proteins. Based on these data we propose that the incorporation of at least some ribosomal proteins into ribosomes is promoted by specific assembly factors. The function of such factors could involve the protection of ribosomal proteins from aggregation, their import into the nucleus and the correct targeting to the rRNA. The aim of this project is the identification of common principles and mechanisms in the assembly path of ribosomal proteins. For this purpose, we will screen for novel assembly factors of small subunit ribosomal proteins. The identified factors will be further characterized with respect to the functions they exert on their ribosomal protein binding partners. To achieve these goals, a combination of biochemical, cell biological and genetic approaches will be employed. So far, little is known about non-ribosomal factors interacting with ribosomal proteins before their assembly with the rRNA. Hence, this project will be an important contribution to our understanding of the early steps in ribosome synthesis. Notably, the ribosome biogenesis pathway is conserved between yeast and humans. Dysregulation of ribosome biogenesis in humans due to altered expression or mutation of ribosomal proteins has been associated with hereditary bone marrow diseases and cancer. Consequently, the understanding of the fundamental question of how ribosomal proteins are assembled into ribosomal subunits will on a long term be important for the mechanistic understanding and the treatment of human diseases.

Ribosomes are the cellular factories responsible for the production of all proteins. When a cell divides, it has to duplicate its complete population of ribosomes (several 100,000s), therefore the synthesis of ribosomes is a central process in all growing cells. Ribosomes are composed of two types of building blocks, ribosomal RNA (rRNA) and ribosomal proteins. These constituents are generated in two different cellular compartments. While rRNA is produced in the nucleus, ribosomal proteins are synthesized in the cytoplasm and need to be transported into the nucleus, where they bind to the rRNA. To allow for a fast and efficient ribosome synthesis process, specialized factors provide assistance and ensure a quick and safe delivery of r-proteins to the nucleus. Import factors mediate the transit of ribosomal proteins into the nucleus. In addition, specific "bodyguards", so called "chaperones" might be needed to keep ribosomal proteins stable and protect them from false interactions. In this project, we aimed at identifying so far unknown chaperones and import factors for ribosomal proteins. In the course of the project, we purified ~30 different ribosomal proteins and identified all bound partners by quantitative mass spectrometry. Using this method, we discovered several so far uncharacterized proteins that bind specifically to only one of the tested ribosomal proteins, suggesting they are specific chaperones for the respective ribosomal proteins. In addition, we found that different ribosomal proteins are bound by different sets of importins. Our discoveries provide a great starting point to gain a deeper understanding how ribosomal proteins are imported and how they are protected by chaperones, thereby ensuring optimal efficiency of ribosome synthesis.Apart from functioning as structural components of ribosomes, some ribosomal proteins additionally also participate in other cellular processes. These "extraribosomal" functions of ribosomal proteins may help the cell to coordinate protein synthesis with other cellular processes. In a second part of the project, we discovered that in human cells, ribosomal protein Rps3 binds to I?B, an inhibitor of the major cellular immune response regulator NF-?B. These results might indicate a link between ribosome synthesis and cellular regulation processes.