Investigating nucleolar dynamics in karyotypically normal human cells by fluorescent protein tagging of genes encoding key nucleolar proteins

The nucleolus, the site of ribosome biogenesis, is the largest membrane-less structure or organelle in the eukaryotic nucleus. It forms around arrays of ribosomal gene (rDNA) repeats termed nucleolar organizer regions (NORs). In humans, NORs are located on short (p) arms of the five acrocentric chromosomes, and are transcribed by RNA polymerase I to generate pre-ribosomal RNA (pre-rRNA) that are subsequently processed and assembled into ribosomes. Nucleoli comprise a fibrillar component (FC), a dense fibrillar component (DFC) and a granular component (GC) as compartments that reflect the sequential steps of ribosome biogenesis, rDNA transcription, pre-rRNA processing and pre-ribosome assembly respectively. RNA Polymerase I (Pol I) rDNA transcription, is the first step for ribosomal assembly, and it is evolutionarily conserved and is generally considered to be one of the most energy consuming process required for cell growth, proliferation, and homeostasis. Sequences surrounding NORs on acrocentric p-arms contribute to the formation of peri-nucleolar heterochromatin, observed in most human cells. In recent years, liquid like behaviour and liquid-liquid phase separation (LLPS) have been proposed to explain both nucleolar formation and internal organisation. A further refined model of nucleoli as comprising multiphase condensates has been derived from in vitro experiments with only a few selected recombinant fusion proteins and in vivo experiments with over expressed fusion proteins in transformed cell lines. In this thesis I develop a strategy for efficient fluorescent protein and Halo-tagging of multiple genes encoding key nucleolar components. The cell model I use, hTert-RPE1, is karyotypically normal, has well characterised NORs and intact cell-cycle check points. Like other non-transformed human cell lines, hTert-RPE1 were previously shown to be highly refractile to CRISPR gene tagging. This is due to low rates of homology directed repair of CAS9 induced DNA double strand breaks1,2 . The key advance is to link targeted integration with selection through the use of viral P2A sequences. The cells lines I have developed provide a platform for future exploration of nucleolar dynamics in live ‘normal’ human cells. Exploitation of these cell models during the later stages of my thesis work was profoundly compromised by the emerging COVID-19 pandemic. Nevertheless, preliminary experiments confirm their utility for investigating nucleolar breakdown and reformation before and after cell division respectively. Additionally, they will provide powerful tools for studying the dramatic reorganisation associated with nucleolar stress. Finally, the strategies and arrays of targeting cassettes I have developed will power future research into other aspects of this critical and most intriguing of nuclear organelles.

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NUI Galway

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Attribution-NonCommercial-NoDerivs 3.0 Ireland
CC BY-NC-ND 3.0 IE

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University of Galway Theses (PhD Theses)