Formation and maturation of the eukaryotic 60S ribosomal subunit

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Ribosome biogenesis comprises, transcription, modification, folding and processing of the rRNA coupled with the incorporation of 80 ribosomal proteins to form the small (40S) and large (60S) subunit. By combining cell biological, biochemical and structural methods in the model yeast Saccharomyces cerevisiae, we gained insight into various steps during 60S ribosome formation. During the next funding period, we will continue our structural and functional studies on this most complicated maturation pathway. The first focus will be on early nucleolar pre-60S particles and the role of several essential proteins belonging to the Brix domain family (Ssf1, Brx1, Rpf1) for initiating 60S assembly. In a second project, we will study the assembly of the 5S RNP into the pre-60S ribosome and how structural maturation occurs with the focus on the Rpf2-Rrs1 complex and a novel assembly factor Cgr1. In a third project, we want to analyze how the catalytic center of the 60S subunit, where peptide formation occurs (peptidyl transferase center or PTC), is formed during 60S biogenesis, which also involves factor removal from this region. Specifically, we will focus on the dismantling AAA ATPase Drg1, and whether and how dominant-negative drg1 mutants affect Nog1 and Nsa2 release from the pre-ribosome. Finally, upon studying Rpl4 incorporation into the pre-60S ribosome, we came across a specific rpl4 mutation that lines the exit tunnel, which caused a ribosome maturation block at a very late step, presumably in the cytoplasm. Hence, we would like to clarify the molecular details of this quality control sensing the intactness of the exit tunnel. From all these planned studies, we expect mechanistic insight into 60S subunit biogenesis, which could also impact our understanding of ribosome-associated diseases.