Characterisation of human nucleolar organisers located in previously uncharted regions of the human genome

Abstract

The nucleolus is a large, sub-nuclear domain and the site of ribosome biogenesis. Nucleoli form around chromosomal loci called ‘nucleolar organiser regions (NORs) located on the short arms of the human acrocentric chromosomes’. NORs house ribosomal RNA genes (rDNA) that are transcribed by RNA polymerase I to generate pre-ribosomal RNA (pre-rRNA) that is subsequently processed and assembled into ribosomes. The nucleolar structure reflects the sequential process of rDNA transcription, rRNA processing and pre-ribosome assembly. The short-arms of the five acrocentric chromosomes are missing from the most recent draft of the human genome and have never been fully sequenced. Evidence thus far suggests sequences proximal and distal to NORs are shared across each acrocentric chromosome. The sequences on the telomeric side of the rDNA are known as the distal junction (DJ). During interphase, DJ sequences are located at the peri-nucleolar heterochromatin, adjacent to the linked rDNA within the nucleolar interior. Despite the heterochromatic environment in which the DJ resides, it has been shown to have a complex sequence and chromatin environment indicative of an actively transcribed functional genomic locus. Two DJ-derived RNA polymerase II transcripts have been identified. They are both spliced and poly-adenylated and have been proposed as long non-coding RNAs function at or near their site of synthesis. In this thesis, I describe a novel, multi-faceted approach to identify and characterise the DJ regions of individual acrocentric chromosomes. Sequence-capture technology in conjunction with mono-chromosomal somatic cell hybrids containing individual acrocentric chromosomes in isolation was used to generate libraries of rDNA-distal sequences for third generation sequencing. Genome editing technology and antisense oligonucleotides were also employed to manipulate and deplete these DJ regions and their transcripts in order to ascertain a function for DJ sequences in NOR behaviour and nucleolar biology. XIV The results obtained during this project show that DJ regions are highly shared across each acrocentric chromosome, suggesting an important role for maintaining NORs in a particular chromosomal context. Slight differences in DJ sequence composition and more extreme differences in further distal sequences between different acrocentric chromosomes suggest possible recombination between nonhomologous acrocentric chromosomes. The two RNA polymerase II transcripts produced by the DJ appear to be essential for normal cell growth. Furthermore, by performing genome-editing experimemnts on the DJ in human cells, I observed cells exhibiting a perturbed nucleolar fusion, hinting at a potential role for these sequences in cell growth, nucleolar function and ribosome biogenesis.