Student Theses and Dissertations

Date of Award

2022

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Klinge Laboratory

Abstract

The ribosome is the RNA-protein machine responsible for the essential task of translating mRNA into proteins. Ribosomes are heterodimers made up of a small subunit (SSU, 40S) and a large subunit (LSU, 60S). At the interface of these subunits, the mRNA is decoded by the small subunit and peptide bond formation is catalyzed by the rRNA of the large subunit. The cell therefore requires the timely and accurate assembly of functional ribosomal subunits, a complex process termed ribosome biogenesis. In addition to the ribosomal RNAs and ribosomal proteins that make up the mature subunits, eukaryotic cells require over 200 trans-acting factors to assemble ribosomes. This process begins in the nucleolus- a subcompartment of the nucleus- where the ribosomal DNA is transcribed to produce pre-ribosomal RNAs (pre-rRNAs). The newly transcribed pre-rRNAs recruit many assembly factors essential to their early folding, but many of these factors exact roles were largely unknown at the start of this work. Additionally, as the rRNA forms the active centers of the ribosome, the initial steps of rRNA folding and chaperoning are of great interest. Structural biology studies have elucidated snapshots of later stages of assembly, but structural understanding of the very early stages of ribosome assembly were limited at the start of this work, and therefore our understanding of early assembly factor function and rRNA folding was restricted. To gain insight into the earliest stages of large subunit assembly, we aimed to isolate and structurally characterize an early nucleolar stage of large subunit assembly. The structures of the nucleolar pre-60S provided insights into the roles of many nucleolus-specific assembly factors. Additionally, we observed that the pre-60S rRNA is largely disordered at this stage in assembly. The rRNA domains making up the solvent exposed side of the LSU are in a nearmature conformation, while the rRNA domains that will eventually form the active centers are largely disordered and prevented from folding (Chapter 2). Assembly factors enforce the open architecture of the rRNA while also preventing premature recruitment of later assembly factors. Initial findings towards the study of an even earlier stage of LSU assembly are also described here (Chapter 4). Among the hundreds of assembly factors are several essential RNA helicases. Their functions and RNA targets are a particularly poorly understood aspect of ribosome biogenesis. One of the most well studied helicases necessary for small subunit assembly is Dhr1. Dhr1 is responsible for removing the U3 small nucleolar RNA (U3 snoRNA) from one of the earliest stable precursors of small subunit assembly called the SSU processome. Structures of the SSU processome revealed that the U3 snoRNA is a key architectural feature of this assembly intermediate. Basepairing between the U3 snoRNA and the small subunit rRNA prevents folding between the subdomains of the SSU rRNA, ensuring they mature separately at this stage of assembly. Characterization of Dhr1 and its co-activator Utp14 in biochemical and structural studies has laid the foundation for understanding how the activity of this key enzyme is regulated (Chapter 3). Overall, this work has contributed to the understanding of nucleolar stages of both small and large subunit biogenesis. The studies of the nucleolar pre-60S have elucidated the structures of several nucleolar specific assembly factors and revealed the architecture of early LSU rRNA folding. Biochemical and structural characterization of Dhr1 provided insight into how this RNA helicase is regulated in small subunit biogenesis. Together, these works have expanded our knowledge of the high level of control the cell exhibits over RNA folding and enzyme activity during the earliest stages of ribosome assembly.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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