Pre-ribosomes Release from the Nucleolus when They Lose Trans Interactions Through Changes in Protein Composition and rRNA Compaction
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.
- Biological Sciences
- Doctor of Philosophy (PhD)