Talk abstracts

Talk on Friday 04:00-04:15pm submitted by Michael Gamalinda

The Yeast 60S Subunit is Structured by Ribosomal Proteins in a Stepwise Fashion

Michael Gamalinda (Carnegie Mellon University), Jelena Jakovljevic (Carnegie Mellon University), Jan Linnemann (Universitat Regensburg), Reyes Babiano, Jesus de la Cruz (Universidad de Sevilla), Philipp Milkereit (Universitat Regensburg), John Woolford, Jr. (Carnegie Mellon University)

Abstract:
Eukaryotic ribosome biogenesis involves regulated processing and folding of precursor rRNAs (pre-rRNAs) and concomitant assembly of ribosomal proteins (r-proteins). Over 200 trans-acting factors are known to be required for pre-rRNA processing, association of proteins, and transit of preribosomes from the nucleolus to the cytoplasm. Recently, it has become more evident that mutations in r-proteins result in a variety of human diseases. In vitro reconstitution studies in bacteria have revealed the hierarchical and cooperative nature of assembly characterized by a sequence of conformational changes in pre-rRNPs and association of r-proteins. However, such studies could not reveal important facets of ribosome synthesis in vivo. We have systematically investigated the roles of thirty-one r-proteins in the synthesis of Saccharomyces cerevisiae 60S subunits by generating yeast strains conditionally expressing each one of these proteins. In vivo depletion of individual r-proteins caused defects in large subunit assembly. Interestingly, we found that these r-proteins fall into broad phenotypic categories: early (processing of 27SA to 27SB pre-rRNA), middle (conversion of 27SB to 7S and 25.5S pre-rRNAs), and late (final maturation into 5.8S and 25S rRNAs ) steps in pre-rRNA processing. Surprisingly, these classes correlate with location of the proteins in mature ribosomes. We examined in further detail two (rpL7 and rpL8) and three r-proteins (rpL17, rpL35, and rpL37) from the early and middle classes, respectively. In the absence of these proteins, preribosomal particles are largely stable. However, when either rpL7 or rpL8 is depleted, assembly factors required for 27SA₃ pre-rRNA processing cannot associate into preribosomes. On the other hand, key assembly factors required for cleavage at the C₂ site fail to join preribosomes lacking rpL17, rpL35, or rpL37.

Keywords: ribosome assembly, ribosomal proteins, pre-rRNA processing