Hierarchical Association of Subcomplexes Drives pre-rRNA Folding during Ribosome Biogenesis in <i>Saccharomyces cerevisiae</i>

<p>Construction of eukaryotic ribosomes requires at least 180 assembly factors. These proteins are thought to drive forward the progressive folding and processing of pre-rRNAs, and binding of ribosomal proteins. Initial characterizations have revealed in which steps of pre-rRNA processing most of these factors function, but only recently have investigations begun to understand how each of these proteins contributes to folding of the nascent rRNA.</p> <p>Work from our lab, part of which is presented in this thesis, has established an association hierarchy between 12 assembly factors and ribosomal proteins necessary for processing 27SA₃ pre-rRNA. The most up-stream factors in this pathway are present in a subcomplex of four assembly factors and two ribosomal proteins. I focused on the understanding the roles of two of these proteins, Nop12 and Pwp1, in ribosome biogenesis. I investigated their timing of association with pre-ribosomes, their role in recruiting other proteins, and the effects on pre-rRNA folding in their absence. By doing so, I show that the effects observed on pre-rRNA processing in the absence of Nop12 and Pwp1 are not simply due to pre-ribosomes lacking a complete inventory of proteins, but rather due to a failure to properly fold the rRNA.</p> <p>I also investigated the function of the DEAD-box protein Drs1 in ribosome biogenesis and show that it functions in two consecutive steps of ribosome assembly. Furthermore, physical and genetic interactions reveal that Drs1 is recruited to pre- ribosomes by a subcomplex of assembly factors that function in the same step of biogenesis.</p> <p>Most importantly, in the course of investigating Drs1, I found that disruption of ribosome biogenesis results in a shift from pre-rRNAs being processed co-transcriptionally, to being processed post-transcriptionally. When this occurs, there is a breakdown of the global hierarchy of ribosome biogenesis. I show that normally late associating proteins associate with pre-ribosomes early in the biogenesis pathway, prior to any pre-rRNA processing steps occurring. Furthermore, I show this shift from co- to post-transcriptional processing is related to cell growth rates, and pre-rRNAs processed post-transcriptionally proceed down an alternative maturation pathway. These results have drastic implications for how we view the overall hierarchy of ribosome biogenesis.</p>