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Title: A "splicing switch" provides dual layers of regulation of laminin alpha 3 : implications for wound healing and squamous cell carcinoma
Author: Troughton, L. D.
ISNI:       0000 0004 7656 8866
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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The laminin α3 gene (LAMA3) generates two major isoforms through alternative promoter usage; LAMA3A and LAMA3B. The ratio between these isoforms has been implicated in squamous cell carcinoma (SCC), with an increased LAMA3A:LAMA3B correlating with poor patient survival. Moreover, the proteins produced, laminin α3a and α3b, have been shown to be functionally distinct in terms of their relative abilities to support adhesion and migration. Two additional LAMA3B promoter-driven transcripts were further identified, LAMA3LN1 and LAMA3LN2; at least one of which is translated into a functional protein, LaNt α31. The additional isoforms are generated through a rare form of alternative splicing called intron retention and alternative polyadenylation (IRPA). As such, IRPA can reduce the proportion of the full-length transcript being produced from a gene. We hypothesised that the IRPA events in LAMA3 contribute to LAMA3B regulation and, moreover, the protein produced can influence laminin-regulated cell behaviour, such as adhesion and migration. Isoform specific qRT-PCR revealed that IRPA is dynamically regulated; and at times, under certain conditions, accounts for up to 50% of all transcripts generated from the LAMA3B promoter. Using a dual colour reporter minigene the mechanisms that regulate the production of one of these isoform were investigated. These studies identified that a weak, but not absent, donor and acceptor splice site are essential for IRPA to occur, and that multiple RNA binding proteins are also likely playing an important role in regulating IRPA. At the protein level, we generated mouse monoclonal Abs specific to LaNt α31, and investigated the expression across the ocular anterior chamber surface epithelium. Interestingly, this revealed LaNt α31 to be restricted to the basal layer of corneal epithelium with low levels of expression, but enriched in the limbus and conjunctival epithelium, where localisation mirrors that of limbal stem cell-associated proteins. In ex-vivo porcine corneal wound and human limbal explant models, LaNt α31 expression was upregulated during the initial phases of re-epithelialisation and in activated limbal regions, but was re-distributed to the basal epithelium once the wound had fully healed. In cultured epithelial cells, expression of eGFP tagged LaNt α31 led to an increase in cell spreading and a reduction in migration speed. In live analyses of new matrix synthesis, LaNt α31 and LMα3 co-distributed and were deposited together, while immunoprecipitation indicated complex formation. Indirect-immunofluorescence revealed changes to LM332 organisation, where tight clusters were observed; with similar findings observed in the distribution of β4 integrin. Interestingly, these changes correlated with an increase in collagen XVII recruitment to the clustered β4 integrin and an increase in LMα3 processing; together pointing to early hemidesmosome maturation. Using novel rabbit polyclonal Abs raised against a LMα3b specific peptide, expression of LMα3b was compare to LaNt α31 in head and neck SCC. Interestingly, a reduction in LMα3b expression in tongue and larynx SCC correlated with a large upregulation of LaNt α31 in most cases, suggestive of a "splicing switch" in. To investigate the consequences of this up-regulation, LaNt α31 expression was induced in the breast adenocarcinoma cell lines MCF-7 and MDA-231. Interestingly, LaNt α31 up-regulation did not cause a migration or invasion phenotype on collagen I, but it did influence the way in which MDA-231 cells invade into matrigel. Specifically, LaNt α31 expressing MDA-231 cells invaded less into matrigel, but interestingly, exhibited single cell invasion as opposed to the multi-cellular streaming observed with controls, suggestive of a LM-specific effect. Together, these data reveal two new ways in which laminin α3 is regulated by IRPA; influencing LAMA3B abundance at the transcript level and influencing laminin organisation at the protein level. These findings have implications for wound healing and SCC.
Supervisor: Hamill, Kevin J. ; Willoughby, Colin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral