An integrative approach of ribosome biogenesis mechanisms in human cells – RIBOMAN

The ribosome is arguably one of the most ancient molecular machines given its universal function in protein synthesis. While its overall structure is remarkably conserved across the three kingdoms of life, ribosome production in eukaryotes has evolved into a more complex assembly process, which includes additional ribosomal proteins (RPs) and accessory factors, along with supplemental RNA extension domains. Defects in ribosome synthesis are now considered pathogenic in a growing family of genetic diseases or acquired syndromes, termed ribosomopathies. Ribosome biogenesis defects have been associated to human genetic diseases such as Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome or the Treacher-Collins syndrome. The cellular stress resulting from defective ribosome biogenesis triggers response pathways, including p53 activation and cell cycle arrest, which appear to be fatal to some physiological processes. 5q- myelodyplasia or a familial form of colon cancer were also associated to haploinsufficiency of RPs. This calls for a deeper understanding of ribosome biogenesis mechanisms in human cells.
Our knowledge of ribosome biogenesis in mammalian cells has lagged behind a wealth of studies performed in yeast. However, advances in gene expression technologies in mammalian cells have recently unraveled substantial differences in the mechanisms of mammalian pre-ribosomal RNA (pre- rRNA) processing and pre-ribosome dynamics. Hence, the Gleizes group has described both new intermediate in the ribosomal RNA (rRNA) maturation pathways and differences in the nucleolytic machineries. Along the same line, the Kutay group has developed a genome-wide siRNA screen that has led to the inventory of human ribosome biogenesis factors. While many pre-ribosomal factors appear to have conserved functions, the dynamics of their association with pre-ribosomes are different in human and yeast, suggesting changes in the spatial coordination of pre-ribosomal assembly. These significant mechanistic differences are likely linked, at least in part, to the emergence of new regulatory pathways in metazoans during evolution.
The time is ripe for exploring in depth these new mechanisms. Here, we propose to: (1) determine the structure of human pre-ribosomal particles at different maturation stages by cryo-electron microscopy (cryo-EM) combined to X-ray crystallography and chemical probing analyses; (2) functionally characterize mechanisms specific to human pre-40S particle formation related to pre-rRNA processing and protein assembly. The groups involved in this proposal possess the tools, expertise and experience that place them in an excellent position to fulfill the ambitious targets of the project. We expect this work to yield major insights into the specific mechanisms of ribosome synthesis in human, but also to broaden our understanding of the relationships between ribosome biogenesis and cellular physiology.