Characterisation of human nucleolar organisers located in previously uncharted regions of the human genome
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2019-01-18Author
Ó Gailín, Michael
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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.