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Subject: search.filters.subject.Non-coding RNA

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    Untangling the interaction between HP1α and the TERRA G-quadruplex : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Roach, Ruby
    Over 50 years ago the DNA double-helix structure was solved, revealing the code in which life is written. This set of instructions equates to three billion base-pairs, amounting to two metres in length, fitting into a human nucleus just six microns across. The stable yet dynamic structure that allows for the functional organisation of the genome is chromatin: a complex formed between DNA and proteins. Chromatin is delineated into two microscopically defined compartments: the less dense gene-rich euchromatin and the structurally compact gene-poor heterochromatin. Protective heterochromatin is constitutively maintained for telomere protection, chromosome segregation, DNA repair, and suppression of transposon activity. Essential for the propagation and maintenance of heterochromatin is Heterochromatin Protein 1α (HP1α), which is comprised of a chromodomain and chromoshadow domain separated by a disordered hinge. The histone code model proposes that HP1α is recruited to regions of heterochromatin by its chromodomain-mediated recognition of silent heterochromatin mark trimethylated lysine 9 of histone H3 (H3K9me3); however, this does not account for the specificity of HP1 paralogs, location-specific recruitment, or the contribution of RNA to heterochromatin formation. Here, Telomeric Repeat-containing RNA (TERRA), transcribed from the telomeres and shown to be involved in telomeric maintenance and stability, is investigated in its interaction with HP1α. The interaction with TERRA is proposed as a means for recruitment of HP1α to telomeres for chromosome end protection. Due to its guanine (G)-rich sequence, TERRA folds into a G quadruplex (G4), a topology distinctly different from canonical nucleic acid structures. The interaction between HP1α and TERRA is therefore investigated to establish the means of interaction between HP1α and a G4, and to examine the HP1α specificity towards TERRA G4s. This work showed that binding to TERRA is dependent upon multiple positively charged patches within the disordered hinge of HP1α, and is also affected by mimicking N-terminal phosphorylation, which alters the structure of HP1α. While the hinge of HP1α binds to a myriad of nucleic acid structures, the globular chromoshadow domain provided the specificity for the parallel G4 topology evident in TERRA. Solution structures of HP1α in complex with TERRA show that HP1α undergoes a conformational shift, becoming less flexible. These results show that noncanonical nucleic acid structures such as those formed by TERRA may act as determinants of HP1α function, serving as signposts in the genome for formation of protective heterochromatin. This biophysical study also further implicates non-canonical structures such as G4s as formidable regulators of genomic function.
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    Characterising a biologically relevant protein-G4 interaction : HP1α and TERRA : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Roach, Ruby
    Our genetic material is intricately folded and protected through the formation of a compact nucleoprotein complex, termed heterochromatin. In addition to controlling the expression of genes, heterochromatin formation is important for the structural integrity of our genome, specifically for the centromeres, the central attachment point of our chromosomes, and also the telomeres, the ends of our chromosomes. The way in which the heterochromatin in these areas is formed and maintained is though the recruitment and binding of the pivotal chromatin regulator, Heterochromatin Protein 1α (HP1α). The current model that explains how and why HP1α is recruited to, and maintained at, regions of constitutive heterochromatin is simple: HP1α binds post-translational modifications on histones (eg. H3K9me3). However, this binding is not high affinity, therefore may not be the sole determinant in HP1α localisation. At the centromeres, it has been shown that a long non-coding RNA transcribed from the peri-centromeres is responsible for recruiting HP1α to this crucial region. At the telomeres, it is proposed that the long non-coding RNA transcribed from the telomeric DNA is responsible for this same purpose. Because of its guanine-rich sequence, it is able to form a non-canonical nucleic acid structure, the G-quadruplex, which may provide the specificity for heterochromatin formation at telomeres. This TElomeric Repeat-containing RNA (TERRA) has been implicated in telomeric elongation pathways, relating it to the immortalisation of cancer cells. It was found that HP1α can specifically recognise the parallel topology of TERRA, binding with high affinity through HP1α’s unstructured hinge. HP1α was also shown to bind other G-quadruplexes of parallel topology, specifically DNA present in promoter and regulatory regions of many proto-oncogenes, implicating HP1α in potential G-quadruplex-dependent gene expression regulatory mechanisms. The interaction shown here between HP1α and TERRA shows a novel mechanism of telomeric heterochromatin formation, providing crucial insights into telomere maintenance and health in human cells, and furthering our understanding of the role of G-quadruplexes in the epigenome.
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    Capture-seq and small RNA-seq to identify noncoding RNAs in the mouse ribosomal RNA gene repeat intergenic spacer : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University (Albany), New Zealand
    (Massey University, 2018) Fitch, Jessica Leigh
    Cancer is a leading cause of mortality in developed countries. Due to the genetic and epigenetic heterogeneity of this disease, we still don’t have effective long-term therapies for many cancers. A characteristic of many cancer cells is an alteration in the structure of the nucleolus - the primary location of the ribosomal DNA (rDNA). The rDNA encodes ribosomal RNA, which is the major structural and catalytic component of ribosomes – the cellular machinery responsible for protein biosynthesis. Accordingly, the rDNA and its transcription is a key regulator of cell proliferation. Despite this critical role, the highly repetitive nature of the rDNA has made it difficult to study, thus it remains an attractive target for anti-cancer therapies. Indeed, the promising anti-cancer drug, CX-5461, developed by our collaborators, targets the rDNA through the inhibition of the rDNA dedicated RNA polymerase I (currently in clinical trials). In preliminary experimentation, there is a dramatic change in expression of non-coding RNAs (ncRNAs) from the rDNA during the transition to malignancy. Although the function of rDNA ncRNAs is almost entirely unknown, ncRNAs from other regions of the genome have a multitude of regulatory functions, including involvement in cancer. We hypothesise that these transcripts play a role in malignancy and CX-5461 sensitivity. Utilising a mouse B-lymphoma model (Eμ-myc), we first applied a high throughput hybridisation-based RNA-sequencing approach (capture-seq), to enrich for rDNA intergenic spacer (IGS) ncRNA transcripts within 11 cDNA sequencing libraries. Regions of transcription throughout the IGS were identified using several bioinformatic tools, and qPCR was performed to validate transcription status as well as assess for CX-5461-dependent transcriptional changes. We also utilised other bioinformatics tools, to predict small RNAs arising from the IGS and other regions of the Eμ-myc genome, and briefly assessed their response to CX-5461 treatment. miRNAs of interest were assessed for potential pathway targets using several bioinformatic targets. Lastly, we aimed to further characterise the Eμ-myc model. With this, we assessed efficacy of methods that could be used for downstream knockdown/over expression analysis. Overall, using the capture-seq method we identified 8 major clusters of exons (known as exon cluster groups), that were consistently predicted between RNA library preparations. These were confirmed to be transcriptionally active by qPCR, with one of these clusters. Additionally, we identified several sites in the mouse rDNA IGS that may express small RNAs, with small RNA reads aligning to these sites with some consistency between library preparations. Some of these, due to presence and absence patterns in either CX-5461 treated or control libraries, may show some signs of treatment-dependent differential expression. We also identified miRNAs from other regions of the genome which show similar patterns. We assessed potential small RNAs for gene target enrichment. No pathways/cellular components appeared to be biologically significant. We assessed a method of viral-mediated gene knockdown in a number of cell lines, which did not show efficacy in the mouse lines we had available. In conclusion, if these exons produce ncRNAs that contribute to malignancy, the ncRNAs will form attractive new targets for therapy, independently or in combination with CX-5461, and could be used as diagnostic and prognostic markers of cancer. The future trajectories of this project include selecting promising IGS transcripts, particularly those differentially expressed, to confirm their size by northern blot. Then, to assess their role in malignant cells, to perform knockdown/overexpression assays and assess cellular response. Further, we would target the rDNA ncRNAs in several cancer and non-cancer cell lines, to broaden our understanding of anti-cancer application.
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    MicroRNA and mRNA analysis of two species of New Zealand Pachycladon : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Carr, Louise Michelle
    MicroRNAs (miRNAs) are small, non-coding RNAs important in post-transcriptional regulation. In this study, potential miRNAs from two New Zealand Pachycladon species, P. cheesemanii and P. fastigiatum, are identified and compared. Sixteen miRNAs were differentially expressed between the species, most of which have roles in flower and leaf development. Potential targets for 15 miRNAs were located in expressed sequence tag (EST) libraries for P. cheesemanii and/or P. fastigiatum, including a new potential relationship in P. cheesemanii between miR825 and MYB29 (AT5G07690), a transcription factor involved in the synthesis of methionine-derived glucosinolates. From the results of the differential expression analysis and target identification, 27 miRNAs from 21 miRNA families were chosen for pre-miRNA sequencing. Sequences of 15 P. cheesemanii miRNA hairpins and 13 P. fastigiatum miRNA hairpins were validated experimentally. Additionally, mRNA-Seq data obtained at the same time as the miRNAs were analysed. A gene ontology analysis indicated enriched terms for defence responses and miRNAs in P. fastigiatum. This study is the first investigation of the miRNAs present in Pachycladon and how their differential expression contributes to the adaptive divergence between the species.
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