Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.773760
Title: Regulation of splicing integrin ɑ6 during development and differentiation
Author: Alshehri, Mohammed Ali
ISNI:       0000 0004 7961 0038
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2017
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Abstract:
Alternative splicing is an important mechanism for creating protein diversity. Integrins are significant in many aspects of cell biology, including cell signalling and interaction with the cell matrix. ITGA6 has two different cytoplasmic C-termini (a6A and a6B) that shift 100% between stem cells and fibroblasts. The primary aim in this thesis was to monitor splicing patterns during development and differentiation integrin subunit alpha 6 (ITGA6) to see which alternative splicing events are similarly regulated in fish and humans using early zebrafish development. The a6A and a6B integrins had been differentially implicated in the expression in the function of breast cancer and cancer stem cells. Therefore, the second aim was to monitor splicing patterns for ITGA6 in different cancer cell lines and to compare them with stem cell patterns, fibroblast, and zebrafish, determining which splicing regulator protein regulates the ITGA6 alternative exon. It was confirmed that the ITGA6 alternative exon 25 was activated by MBNL1, RBFOX2 and ESRP in cancer cell lines, and PTBP was discovered as a novel regulator for ITGA6 splicing that inhibited the exon of ITGA6 in cancer cell line. The third aim for this project was to identify the mechanism of splicing of this ITGA6 alternative exon, including identifying the PTB binding site that regulates ITGA6. A minigene system was established to study the regulation of the ITGA6 alternative exon. The ITGA6 1.3 minigene positively responded to siRNA mediated depletion of splicing factors in the same way as the endogenous gene, indicating this minigene was a good model. The alternative exon of ITGA6 was activated by MBNL1 and was inhibited by PTBP, leading to more production of ITGA6B. Using this minigene plasmid it was confirmed that PTBP inhibited alternative splicing of ITGA6. The last aim of this chapter was to discover the PTB binding sites. Through a series of in silico analyses, a binding site for PTB was identified downstream of the regulated exon. Surprisingly, loss of this PTB binding site actually repressed this splicing event. These data suggest that PTB both activates this alternative splicing event through direct RNA-protein interactions, but also more strongly represses this exon, possibly through protein interactions with other regulatory factors.
Supervisor: Not available Sponsor: Saudi Arabian Culture Bureau in London ; Najran University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.773760  DOI: Not available
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