Date of Award
Doctor of Philosophy (PhD)
John L. Woolford, Jr
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.
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.
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.
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.
Talkish, Jason, "Hierarchical Association of Subcomplexes Drives pre-rRNA Folding during Ribosome Biogenesis in Saccharomyces cerevisiae" (2013). Dissertations. 271.