Nucleolar Stages of 60S Subunit Assembly Involve Complex and Dynamic Changes in Pre-rRNA Structure and Pre-Ribosome Protein Composition

2019-02-28T16:17:10Z (GMT) by Stephanie Biedka
Ribosome biogenesis in eukaryotes is a complex process spanning three cellular compartments and requiring the coordinated transcription, processing, modification, and structural remodeling of immature ribosomal RNA (rRNA) and binding of ribosomal proteins (r-proteins). This process is facilitated by protein assembly factors (AFs), which participate in the assembly process in various ways,<br>but are not incorporated into the mature ribosome. The functions of most of the Saccharomyces cerevisiae r-proteins and AFs in biogenesis of the large ribosomal<br>subunit have been studied to some extent. However, for the most part, we still lacked a clear understanding of the step-by-step process of ribosome assembly, and of how r-proteins and AFs participate in this process.<br>In this dissertation I will describe my studies of a group of AFs and r-proteins required for cleavage of 27SB pre-rRNA at the C2 site during nucleolar stages of ribosome biogenesis. The period of 60S subunit assembly preceding C2 cleavage is marked by the entry and exit of numerous AFs, massive changes to pre-rRNA structure, initial stages of functional center construction, and preparation of the<br>pre-ribosome for transit out of the nucleolus.<br>My work with mutants that cannot carry out C2 cleavage, combined with cryo-electron microscopy structures of pre-ribosome assembly intermediates that were published by our lab and others during the course of my studies, have allowed us to determine the order of remodeling events preceding C2 cleavage. Furthermore, these data have allowed us to create models as to how and why these remodeling events occur. My work revealed that in almost every mutant in which C2 cleavage is blocked, ribosome biogenesis is actually blocked several steps upstream of C2 cleavage. Rather than directly participating in C2 cleavage,<br>most proteins previously characterized as being necessary for C2 cleavage appear to be required for association of the AFs Noc3 and Spb1 with pre-ribosomes. Intriguingly, my studies have also revealed that C2 cleavage is not<br>necessary for subsequent stages of ribosome biogenesis. Instead, 60S subunits that have not undergone C2 cleavage are able to engage in translation. This finding<br>was wholly unexpected, as C2 cleavage was long considered an important checkpoint in large ribosomal subunit assembly. I also investigated how the DEAD-box AF Drs1 participates in construction of the large ribosomal subunit. My studies of Drs1 included determining the effects<br>of depleting, inactivating, and truncating this AF. Drs1 is necessary for both early stages of ribosome biogenesis and for the stages of assembly preceding C2 cleavage. My work has yielded models as to how exactly Drs1 facilitates these<br>stages of assembly. Finally, I investigated potential roles of the r-proteins L7 and L20 in rotation of 5S rRNA during nucleoplasmic stages of assembly. This work provided<br>evidence that L7 and L20 have roles in ribosome biogenesis that were not discovered when the effects of depleting these proteins was initially studied. <br>