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dc.contributor.authorSmith, Jeffrey Michael
dc.description© 2021 Jeffrey Michael Smith
dc.description.abstractRNA-binding proteins (RBPs) are classically regarded as facilitators of gene expression. In recent years, however, RNA-protein interactions have also emerged as a pervasive force in the regulation of homeostasis. The compendium of proteins with provable RNA-binding function has swelled from the hundreds to the thousands astride the partnership of MS-based proteomics and RNA Sequencing. At the foundation of these advances is the adaptation of RNA-centric capture methods that extract protein that has been crosslinked in its native environment. These methods reveal snapshots in time displaying an extensive network of regulation and a wealth of data that can be used for both the discovery of RNA-binding function and the molecular interfaces at which these interactions occur. This thesis describes the development of an extraction method that purifies RBP-RNA complexes. This method differentiates itself from other RBP-discovery protocols in that it 1) purifies these complexes so completely that RBP identification can be conducted qualitatively and without differential abundance analysis, 2) permits transcript-targeted capture with sequence-specific oligos, 3) permits global, sequence-agnostic capture 4) both RBP and its bound RNA are isolated intact and 5) can reliably interrogate RBPs at depths that exceed present methods without metabolic or molecular labelling. The performance of this method is first assessed with a census of proteins that directly interact with global, or targeted, RNA transcripts from model cell lines. These efforts are then extended to investigate how protein-RNA interactions change during transition from quiescence to proliferation and then contraction in primary murine CD8+ T cells. Finally, these studies demonstrate how cellular responses provoke different proteins to moonlight as RNA binders and sheds light on a network of complex, co-evolved molecular machines.
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dc.subjectMass Spectrometry
dc.subjectSystems Biology
dc.subjectRNA Binding Proteins
dc.subjectExperimental Methods
dc.titleThe Purification, Identification, and Measurement Of RNA-Binding Proteins
dc.typePhD thesis
melbourne.affiliation.departmentMedical Biology
melbourne.affiliation.facultyMedicine, Dentistry & Health Sciences
melbourne.thesis.supervisornameAndrew Webb
melbourne.contributor.authorSmith, Jeffrey Michael
melbourne.thesis.supervisorothernameMelissa Davis
melbourne.thesis.supervisorothernameAaron Jex
melbourne.tes.fieldofresearch1310109 Proteomics and intermolecular interactions (excl. medical proteomics)
melbourne.tes.fieldofresearch2310299 Bioinformatics and computational biology not elsewhere classified
melbourne.tes.fieldofresearch3310114 Systems biology
melbourne.tes.fieldofresearch4310505 Gene expression (incl. microarray and other genome-wide approaches)
melbourne.accessrightsThis item is embargoed and will be available on 2023-10-26.

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