Characterising the RNA modification and polyadenylation landscape at single molecule resolution using third-generation sequencing technologies

dc.contributor.advisor Mattick, John
dc.contributor.advisor Novoa Pardo, Eva Maria
dc.contributor.advisor Cooper, Antony Begik, Oguzhan 2022-02-07T05:28:37Z 2022-02-07T05:28:37Z 2021
dc.description.abstract RNA modifications, collectively referred to as the ‘epitranscriptome’, are not mere decorations of RNA molecules, but can be dynamically regulated upon environmental queues and changes in cellular conditions. This dynamic behaviour is achieved through the RNA modification machinery, which comprises “writer”, “reader” and “eraser” proteins that modify, recognize and remove the modification, respectively. Chapter1 presents a comprehensive analysis of the RNA modification machinery (readers, writers and erasers) across species, tissues and cancer types, revealing gene duplications during eukaryotic evolution, changes in substrate specificity and tissue- and cancer-specific expression patterns. Chapters 2 and 3 presents the exploration and development of novel methods to map and analyze RNA modifications transcriptome-wide. Nanopore direct-RNA sequencing technology was used to provide RNA modification maps in full-length native RNA molecules. Firstly, it is shown that RNA modifications can be detected in the form of base-calling ‘errors’, thus allowing us to train Support Vector Machine models that can distinguish m6A-modified from unmodified sites, both in vitro and in vivo. Secondly, it is demonstrated that distinct RNA modification types have unique base-calling ‘error’ signatures, allowing us to exploit these signatures to distinguish different RNA modification types. It is found that pseudouridine has one of the most distinct signatures, appearing in the form of C-to-U mismatches. Finally, this information was used to predict novel pseudouridine sites on ncRNAs and mRNAs transcriptome-wide, as well as to obtain quantitative measurements of the stoichiometry of modified sites. Chapter 4 presents the development of a novel nanopore-based method, which is termed ‘Nano3P-seq’, to simultaneously quantify RNA abundance and tail length dynamics in individual molecules in both the coding and non-coding transcriptome, from cDNA reads. It is demonstrated that Nano3P-seq offers a simple approach to study the coding and non-coding transcriptome at single molecule resolution regardless of the tail ends. Together, this work provides a comprehensive framework for the study of RNA modifications and polyA tail dynamics using third generation sequencing technologies, opening novel avenues for future works that aim to characterize their dynamics and biological roles both in health and in disease.
dc.publisher UNSW, Sydney
dc.rights CC BY 4.0
dc.subject.other Nanopore sequencing
dc.subject.other RNA modifications
dc.subject.other PolyA tail length
dc.subject.other Epitranscriptomics
dc.subject.other RNA Dynamics
dc.title Characterising the RNA modification and polyadenylation landscape at single molecule resolution using third-generation sequencing technologies
dc.type Thesis
dcterms.accessRights open access
dcterms.rightsHolder Begik, Oguzhan
dspace.entity.type Publication
unsw.relation.faculty Medicine & Health
unsw.relation.faculty Science Clinical School St Vincents Hospital Clinical School St Vincents Hospital School of Biotechnology & Biomolecular Sciences School of Medical Sciences
unsw.subject.fieldofresearchcode 3101 Biochemistry and cell biology
unsw.subject.fieldofresearchcode 3102 Bioinformatics and computational biology
unsw.thesis.degreetype PhD Doctorate
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