Identifying individual ribosomal RNA gene repeats to investigate their behaviour

Ribosomal RNA forms the major structural and catalytic components of ribosomes. In eukaryotes, the ribosomal RNA genes (rDNA) are present as multiple tandem repeats that are transcribed by a dedicated polymerase, RNA polymerase I. While hundreds of rDNA repeats are present, only a subset is transcribed in a cell at any given time, with the transcriptional status determined epigenetically. Here, I sought to test whether the transcriptional status of individual rDNA repeats is inherited through mitotic cell division in human cells. This required a system to distinguish individual rDNA repeats within a genome. I first attempted to achieve this by tagging each rDNA repeat with a unique “barcoding” DNA sequence using CRISPR-Cas9, first in human cells then in the yeast S. cerevisiae, but this was unsuccessful for both species. Therefore, I instead utilised deep sequencing to identify single nucleotide polymorphisms (SNPs) in the rDNA that enabled me to distinguish rDNA repeats in cultured human cells. I also used RNA-seq to identify expressed rRNA variants in the same cells. This enabled me to identify numerous rDNA SNP variants, with only a subset being expressed. To test if rDNA repeat transcriptional status is inherited, I made two daughter populations by isolating single cells from the population I originally characterized and expanding them. I then sequenced the RNA from these populations and identified SNP rRNA variants. The variants were expressed at remarkably similar frequencies across all populations, suggesting that rDNA repeat transcriptional status is strongly inherited through mitotic cell division. To phase variants to individual rDNA repeats, I performed Cas9-enriched Nanopore sequencing. This enabled me to construct rDNA variant haplotypes and to determine the methylation state of each rDNA repeat. I found that the methylation state of reads containing the various rDNA SNP variants was correlated with the level of expression of those variants, suggesting that rDNA methylation suppresses rRNA expression. Together, these results suggest that the transcriptional status of individual rDNA repeats is faithfully inherited through cell divisions, and that methylation-based silencing of rDNA repeats might be one mechanism by which this inheritance is achieved.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biological Sciences

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2026

Author dc:creator

• Schischka, Megan Ann

Advisors dc:contributor.advisor

• Ganley, Austen
• O'Sullivan, Justin

• Molecular Genetics

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/74819

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/74819