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Title: Roles of R-loops in the Trypanosoma brucei genome and antigenic variation
Author: Briggs, Emma Marie
ISNI:       0000 0004 7654 985X
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2018
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The genome of the eukaryotic parasite Trypanosoma brucei is both dynamic and unconventional in several aspects. In comparison with other eukaryotic genomes, where the majority of protein coding genes are associated with their own transcriptional promoters, T. brucei transcribes almost all protein-coding genes polycistronically. Transcription initiates from broad regions that lack defined promoter sequences and RNA Polymerase II then traverses up to hundreds of genes, generating a pre-mRNA that then requires trans-splicing and polyadenylation to generate mature mRNAs. Termination of transcription, via virtually unknown processes, occurs where two multigene transcription units converges or, in some cases, adjacent to a downstream transcription initiation site. RNA Polymerase II transcribes the majority of protein-coding genes in this manner, negating any differential gene expression via transcriptional control. A further unusual aspect of the genome is the dedication of as much as a third of the coding capacity to elements of antigenic variation. When infecting the mammalian host, parasites express a dense protein coat of variant surface glycoprotein (VSG). In order to evade host immune elements, T. brucei switches expression to antigenically distinct VSGs, employing a repertoire of ~2,000 genes. Both transcriptional and recombination-based strategies enable the parasite to either switch transcription between ~15 expression sites, each housing a distinct VSG, or relocate VSG sequence from silent gene arrays into an active VSG expression site. Although multiple factors have been found to regulate these processes, the events which trigger a VSG switch by either pathway are unclear. R-loops are three stranded structures containing an RNA-DNA hybrid and displaced single-stranded DNA. Although potentially deleterious to genome integrity, R-loops have been linked to transcription initiation and termination, DNA replication and recombination events. In this study, the potential for R-loop involvement in these fundamental genome functions of T. brucei was investigated. Firstly, Ribonuclease (RNase) H enzymes, which resolve the RNA-DNA hybrid portion of R-loops, were characterised, revealing T. brucei expresses potentially three distinct catalytic enzymes, two functioning in the nuclear genome and one in the kinetoplast(mitochondrial) genome. Nuclear RNase H activity was depleted by null mutation or RNAi mediated knockdown of the nuclear RNase H enzymes, showing that while one RNase H, TbRH1, is non-essential, loss of the other, TbRH2, caused several growth and genome integrity defects. As it was hypothesised to increased levels of RNA- DNA hybrids of the genome, RNA-DNA hybrids were mapped in wild type parasites and those lacking RNases H using a specific antiserum, S9.6. This mapping identified the conserved formation of R-loops at centromeres, retrotransposon-associated genes, rRNA and tRNA genes. R-loop enrichment was also uncovered at RNA Polymerase II transcription start sites, as documented in mammalian genomes. DNA damage was specifically increased at these sites after TbRH2 depletion, indicating efficient resolution of these transcription initiation-associated R-loops is critical for genome maintenance. In contrast, R-loops were not associated with DNA replication or transcription termination suggesting RNA-DNA hybrids are not involved in these processes in T. brucei. The most abundant sites of R-loop enrichment were found to be at the nucleosome depleted regions located between the coding regions of polycistronically transcribed genes and are associated with polyadenylation and trans-splicing, highlighting a novel correlation of R-loops with pre-mRNA processing. Lastly, R-loops were mapped to VSG expression sites where their abundance increased after ablation of RNase H activity, an effect that was associated with both increased DNA damage and VSG switching, uncovering an R-loop-driven mechanism of antigenic variation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)