University of Cambridge
Interrogations of protein and RNA: localisation, interactions and dynamics
Abstract
dc:description.abstractEukaryotic cells are characterised by a high degree of structural partitioning into distinct microenvironments dedicated to diverse and specific roles. To maintain these microenvironments, the functions of endogenous RNAs and proteins are fine-tuned by a set of. post-transcriptional and post-translational regulation mechanisms fundamental to complex cellular processes, throughout the stages of development or in response to acute stimuli. Whereas microscopy- or affinity-based methods are routinely employed to determine the location or the interactions of a limited number of proteins or RNAs, a system-wide analysis enables a thorough understanding of the functional and spatial dynamics of the proteome and transcriptome. In this Thesis, two biochemical fractionation-based techniques were developed in the quest for subcellular exploration of the proteome and transcriptome. In addition, two biological questions were investigated with regard to the post-transcriptional control of the ultraviolet irradiation induced stress response and stem cell pluripotency phase transition. In Chapter 3, a one-step subcellular fractionation procedure using multiple iodixanol containing gradients, termed “MultiLOPIT”, is presented, which provides high-resolution organelle resolution for comparative applications in different cell states or conditions. In Chapter 4, characterisation of the dynamics of protein and RNA-binding proteins, post-translational modification, and spatiotemporal localisation are investigated in response to UV-induced cellular stress. This multi-omics approach highlights selective cytosolic translation programmes and chaperone machinery upon UV irradiation. Chapter 5 describes an efficient, high-throughput and scalable method for simultaneous mapping of the subcellular transcriptome, proteome and RBPome. Finally, chapter 6 delineates the dynamics of the post-transcriptional regulatory programmes mediated with RBPs, highlighting ribosome biogenesis and splicing mechanism involvement through the exit of naïve pluripotency. The results of the research in this Thesis offer a strong basis for the application of localisation, interactions and dynamics studies of RNA and proteins, which enable multi-layered system-level interrogations of cell biology.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Elzek, Mohamed
- Advisors dc:contributor.advisor
-
- Lilley, Kathryn
- Willis, Anne
Subjects
dc:subject × 10Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
- 0000-0003-0594-6543
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/319304