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Pre-ribosomes Release from the Nucleolus when They Lose Trans Interactions Through Changes in Protein Composition and rRNA Compaction

thesis
posted on 25.04.2022, 13:57 authored by Amber LaperutaAmber Laperuta

   In eukaryotes, the ribosome assembly pathway proceeds through three cellular  compartments: the nucleolus, followed by the nucleoplasm, and then the cytoplasm. As  nascent ribosomes assemble, precursor rRNAs (pre-rRNAs) are processed, modified,  folded, and compacted as ribosomal proteins (RPs) bind. Along the way, the process is  facilitated by ribosome biogenesis factors (RiBi factors). The functions of many RPs and  RiBi factors in S. cerevisiae ribosome biogenesis have been assessed to some extent.  Recently, the application of cryo-EM technology to studies of ribosome assembly has  rapidly advanced our understanding of the mechanisms of ribosome biogenesis.  However, these structures only provide limited snapshots of the process. Structures are  not enough to discern the mechanisms of ribosome assembly, nor do they explain how  ribosome biogenesis intersects with the intercellular environment.  In this dissertation, I connect the ribosome assembly pathway to the nucleolar  environment by primarily focusing on the stage in ribosome biogenesis where nascent  ribosomal subunits are released from the nucleolus. During this period of assembly, pre?ribosomes proceed from the phase-separated environment of the nucleolus to the less  crowded and biophysically distinct environment of the nucleoplasm. My studies address  two questions: why are nascent ribosomal subunits released from the nucleolus during a  particular stage in their assembly, and how do four proteins that function during this stage  during the assembly of the large ribosomal subunit (RPs uL2 and eL43 and RiBi factors  Puf6 and Nog2) influence their release from the nucleolus.  Because the nucleolus is a phase separated organelle, I studied the ribosome  biogenesis pathway from the perspective of liquid-liquid phase biology. I systematically analyzed all proteins that function during ribosome biogenesis for regions within their  sequences that are predicted to interact in trans. Additionally, I analyzed published cryo?EM models of pre-ribosomes undergoing both nucleolar and post-nucleolar stages of  assembly for pre-rRNA and protein regions that are predicted to interact in trans. My work  reveals six general principles that connect the mechanisms of ribosome biogenesis to the  localization of pre-ribosomes within the nucleolus. I also provide evidence that these  principles are conserved among eukaryotes. This supports and informs the idea that the  probability of nucleolar release increases as pre-ribosomes undergo maturation within the  nucleolus.  My studies on the mechanisms of the nucleolar release of the assembling large  ribosomal subunit identified the most mature, nucleolar assembly intermediate yet  discovered. Furthermore, my work revealed that depletion of uL2, eL43, Puf6, and Nog2  caused incomplete disassociation of RiBi factors from the nascent large subunit and  inhibited the compaction of rRNA. We determined that these events are critical for the  release of pre-ribosomes from the nucleolus. Intriguingly, we found that Nog2 serves a  critical role during the stages of nucleolar release as aberrant association of Nog2 with  late nucleolar intermediates caused them to release from the nucleolus prematurely.  My work is among the first to analyze the release of the large ribosomal subunit  from the nucleolus through two perspectives: liquid-liquid phase separation and ribosome  biogenesis mechanisms. Further, these studies lay the groundwork to investigate the  relationship between ribosome biogenesis mechanisms, nucleolar morphology, and  nucleolar function.   

History

Date

09/12/2021

Degree Type

Dissertation

Department

Biological Sciences

Degree Name

  • Doctor of Philosophy (PhD)

Advisor(s)

John L. Woolford Jr.

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