| Maturation of the large ribosomal subunit (LSU) in eukaryotes is a complex and highly coordinated process that is carried out by a large, dynamic ribonucleoprotein complex, recently termed the LSU processome. Over 90 ribosome biogenesis factors have been shown to dynamically associate with the LSU processome throughout the course of LSU assembly. However, little is known about how these factors interact with each other within the LSU processome. In this study, I took a systems biology approach to interrogate the organization and architecture of the LSU processome. I performed a semi-high-throughput, array-based, directed yeast two-hybrid assay to map the protein-protein interactions of the LSU processome. I assayed ∼4,900 protein-protein interactions and identified 232 high-confidence, binary interactions among the nucleolar LSU processome proteins. The resulting LSU processome interactome represents a 4-fold increase from current knowledge and has served as a springboard for the experiments presented here.;Inspection of the LSU processome interactome revealed significant insight regarding the organization and function of the assembly proteins within the LSU processome. Clustering analysis of the interactome predicted the existence of both previously identified and novel, putative subcomplexes within the LSU processome. The LSU processome interactome also exposed an interaction network among four of the LSU DExD/H-box RNA helicases and revealed the presence of several hub proteins including Nop4, whose human ortholog, RBM28, is mutated in the ribosomopathy ANE syndrome.;Both Nop4 and its human ortholog, RBM28, are comprised of four RNA recognition motifs (RRMs). I demonstrated that the third and fourth RRMs of Nop4 mediate protein-protein interactions and are essential for Nop4's function as a hub. Interestingly, the missense mutation underlying ANE syndrome lies in the third RRM. Therefore, I used Saccharomyces cerevisiae as a model system to investigate the affects of the ANE syndrome mutation on LSU assembly. Introduction of the analogous ANE syndrome mutation into Nop4 impaired Nop4's function as a hub as it abrogated interaction with several of Nop4's protein partners, which ultimately resulted in growth and pre-rRNA processing defects. Thus, in mapping the LSU processome interactome, I have elucidated the molecular basis of ANE syndrome as a defect in protein-protein interactions. |
