Structural and Functional Studies on the Role of Noc3p for Large Ribosomal Subunit Maturation in Saccharomyces cerevisiae

Ribosomes are macromolecular ribonucleoprotein (RNP) particles that catalyze the translation of mRNA into proteins in all living cells in all kingdoms of life. The ongoing production of ribosomes ensures the availability of a sufficient amount of proteins, which are required for various cellular processes. Ribosome biogenesis represents a highly energy consuming and complex process. Starting in the nucleolus, RNA polymerase I and III transcribe the ribosomal RNA (rRNA) encoding genes to generate precursor rRNAs, which undergo a series of processing, folding, modifying and assembly events in the course of maturation. In the yeast Saccharomyces cerevisiae, 79 ribosomal proteins (RPs), four rRNAs, several snoRNAs and more than 150 transiently acting biogenesis factors operate in a concerted and highly dynamic network to generate the mature ribosomal subunits. During the past decades, a lot of effort has been made to elucidate and further understand the synthesis of ribosomes as paradigm of RNP biogenesis. Extensive research on this field has produced detailed characterization of ribosome biogenesis factors like the maturation step for which they are crucial for or the molecular function they exert. Nevertheless, the role of many factors remains further and requires extensive further investigation. The “Noc-proteins” belong to the broad group of biogenesis factors, which have been identified essential for large ribosomal subunit (LSU) maturation (Noc1p/2p/3p). Accompanying studies have indicated the existence of the dimeric complexes Noc1p/Noc2p and Noc2p/Noc3p, which possibly associate with early and intermediate LSU precursor particles, respectively. In this study, focus was laid on the role of Noc3p. Single molecule cryo-EM revealed the association of Noc3p with subsets of different pre-ribosomal populations of intermediate maturation states. Thereby, a time interval for the association of Noc3p with pre-LSU particles could be deduced within a series of nuclear maturation steps. Simultaneously, first structural characterization could be obtained for the position of Noc2p, the Noc3p binding partner. Furthermore, the basis of the time window, in which association of Noc3p with pre-ribosomes has been observed and the prerequisites for its recruitment and release to and off pre-LSUs has been explored. The results revealed that both recruitment and release of Noc3p depends on the pre-ribosomal assembly state. The varying impact of the single ribosomal proteins (r-proteins) has been discussed in regard to their known binding sites in LSU particles. Applying a versatile tethered tertiary structure probing approach allowed investigation of possible structural alterations that impact or postpone the timely related release of Noc3p. Indeed, an impeded release could be backtracked to an altered conformational state of pre-LSU domain III upon knock-down of particular r-proteins. Finally, structure-based mutagenesis of Noc3p helped to reveal sites within the protein that are essential for the formation of the heterodimer with Noc2p as well as for the association with the pre-LSU. Further studies will be necessary to figure out the binding mode of Noc3p to pre-ribosomes and to which extent it might as well rely on the presence of Noc2p. For this, structural information on Noc2p as whole would greatly help to further address this issue. Altogether, the results presented in this work allow to hypothesize towards a role of Noc3p as sensor for LSU biogenesis and provide a solid base for further investigation