Characterisation of the Rrp5p/Noc1p/Noc2p protein complex and its function in ribosome biogenesis of Saccharomyces cerevisiae

Eukaryotic ribosome biogenesis is a very complex process that includes synthesis of the structural components (ribosomal RNAs (rRNAs) and proteins (r-proteins)), processing and folding of rRNA precursors, as well as assembly of the r-proteins onto the rRNA. Ribosome biogenesis starts with the transcription of the genes encoding the rRNAs (rDNA) in the nucleolus by RNA polymerase I and III, and includes transport of pre-ribosomal particles (pre-ribosomes) through the nucleus and export into the cytoplasm, where the final maturation steps occur. In addition to the structural components, these processes require the function of ~75 small nucleolar RNAs and of more than 150 non-ribosomal proteins termed biogenesis factors, which transiently interact with different pre-ribosomes. It could be shown that several subsets of biogenesis factors form protein modules, which are supposed to constitute building blocks of pre-ribosomes and/or to function together in ribosome biogenesis. In this work, a protein complex consisting of the proteins Rrp5p, Noc1p and Noc2p from Saccharomyces cerevisiae could be reconstituted from heterologously expressed proteins. Noc1p and Noc2p are biogenesis factors of the large ribosomal subunit (LSU), whereas Rrp5p is required for maturation of both the large and the small ribosomal subunit (SSU). Analyses of pairwise interactions between the proteins, as well as negative stain electron microscopy of the purified complex provided further insights into architectural and structural features of the Rrp5p/Noc1p/Noc2p biogenesis factor module. Ex vivo purifications of the module components and analyses of co-purified RNAs and proteins indicated that the Rrp5p/Noc1p/Noc2p module is predominantly associated with the first specific pre-LSU particles. In addition, Rrp5p, Noc1p and Noc2p showed association with early, common ribosomal precursor particles, which are formed before the pathways leading to the small and the large ribosomal subunit are separated. Furthermore, the module components co-purified specific regions of rDNA chromatin from cells treated with crosslinking reagents, and Rrp5p and Noc1p were identified as components of chromatin transcribed by RNA polymerase I. Accordingly, the Rrp5p/Noc1p/Noc2p module appeared to be associated with nascent rRNA precursor transcripts, providing further evidence that the module is recruited very early in ribosome biogenesis. Individual inactivation or depletion of Rrp5p, Noc1p or Noc2p in vivo resulted in severely decreased levels of LSU specific pre-rRNA species and the appearance of aberrant pre-rRNA fragments. In addition, analyses of truncated noc1 alleles indicated that impaired interactions of Noc1p with Noc2p, Rrp5p or pre-rRNA result in similar pre-rRNA processing phenotypes, suggesting that in absence of the Rrp5p/Noc1p/Noc2p module pre-ribosomes are destabilised and pre-rRNAs are prone to degradation. Furthermore, in vivo depletion of one module component and subsequent analyses of the association of the respective non-depleted proteins with pre-rRNA indicated a mutually independent binding of Rrp5p and Noc1p/Noc2p to pre-ribosomes. Accordingly, the module most probably has several binding sites on pre-ribosomal particles. In summary, the results presented here suggest that formation of the Rrp5p/Noc1p/Noc2p module plays a role in the structural organisation of early LSU precursor particles and thereby contributes to their stability, possibly by preventing inappropriate access of endo- and exonucleases to pre-rRNA. Besides, potential mechanisms of the Noc1p/Noc2p independent function of Rrp5p in SSU biogenesis, and a model for the recruitment of the Rrp5p/Noc1p/Noc2p module to pre-ribosomes are discussed. Future studies will be required to determine the structure and architecture of this biogenesis factor module in detail. Furthermore, analyses of the RNA binding and folding activities of the module components, and of the impact of the module on the recruitment of r-proteins and/or other biogenesis factors to early pre-ribosomes will help to understand the precise molecular function of the Rrp5p/Noc1p/Noc2p module in ribosome biogenesis. As all three proteins have homologues in higher eukaryotes, it will be interesting to investigate if formation and function of this module are conserved in evolution.