Structural and Biochemical Characterization of snoRNP Assembly Factors

Eukaryotic ribosome is maturated through a complicated process orchestrated by a large network of biogenesis factors. Small nucleolar ribonucleoprotein particles (snoRNPs) contribute to ribosome biogenesis by processing and chemically modifying ribosomal RNA (rRNA). Several snoRNP proteins are found to be unstable and require the chaperone activities of two AAA+ ATPase complexes, R2TP and R2BRH, in collaboration with the heat shock protein, Hsp90. There is currently is no structural information of R2TP and R2BRH and their interactions with snoRNP proteins or Hsp90 (CHAPTER 1). We obtained a structural model of the Saccharomyces cerevisiae (Sc) R2TP complex made up of two AAA+ ATPases, Rvb1/2p, and two Hsp90 co-chaperone proteins, Tah1p and Pih1p, by a combination of analytical ultracentrifugation, chemical cross-linking, and electron cryomicroscopy methods (CHAPTER 2). These studies showed that that the Pih1p-Tah1p heterodimer caps the Rvb1/2p heterohexameric ring through its association with the nucleotide-sensitive insertion domain of Rvb1/2p. Here I characterized how the assembled ScR2TP interacts with a client protein, Nop58p and with Hsp90 and how these interactions depend on nucleotide binding/hydrolysis (CHAPTER 2 and CHAPTER 3). I show that Nop58p binds primarily to Pih1p and upon ATP or ADP binding, dissociates along with Pih1p-Tah1p from R2TP. I also showed that Hsp90 forms a ternary complex with isolated Pih1p-Tah1p but not with R2TP, suggesting two separate chaperone activities for snoRNP proteins. On the basis that Hsp90 is localized to cytosol and R2TP is to nucleus, I propose that snoRNP proteins are separately stabilized by both activities in cytosol and nucleus, respectively. I have begun characterizing Sc R2BRH that comprises Rvb1/2p and three additional proteins, Bcd1p, Rsa1p, and Hit1p. I show that Bcd1p interacts with Rvb1/2p in the presence of ADP and that the Rsa1p-Hit1p heterodimer interacts with another snoRNP protein, Snu13p (CHAPTER 4). The nucleotide-dependent assembly of R2BRH contrasts the nucleotide-independent assembly of R2TP, suggesting that nucleotide binding can coordinate assembly of different snoRNP proteins, thereby ensuring a proper order of snoRNP assembly (CHAPTER 5).