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Title: Post-transcriptional regulation of ALAS1 expression by haem
Author: Redding, Sadie Jane
ISNI:       0000 0004 2740 8735
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2007
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Haem is the prosthetic moiety of numerous haemoproteins critical for the function of all aerobic cells. Its biosynthesis is a tightly controlled process since high intracellular haem concentrations are cytotoxic, whilst haem deficiency impedes the activity of essential haemoproteins. In the liver and probably all other non- erythroid cells, haem supply is regulated primarily through feedback regulation of the stability of the mRNA encoding aminolevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme in the haem biosynthetic pathway. However, the underlying mechanism of this destabilisation is unknown. Consequently, the primary aim of this thesis was to determine how haem regulates ALAS1 mRNA stability to control its own synthesis in non-erythroid cells, using the human hepatoma cell line, HepG2. In humans, the ALAS1 exon lb in the 5'-untranslated region (UTR) is alternatively spliced to produce a minor and major form of the enzyme. This thesis has demonstrated that unlike the major ALAS1 5'-UTR, the minor 5'-UTR causes a downstream heterologous RNA to be poorly translated. In addition, the minor ALAS1 isoform is relatively resistant to haem-mediated decay. Using reporter assays and RT-PCR, we have shown that the human ALAS1 mRNA contains a coding region determinant (CRD) that mediates its haem-sensitivity in HepG2 cells. This CRD can function independently of the ALAS1 5'- and 3'-UTR. Furthermore, this haem-mediated CRD has to be translated to function. RNA- electromobility shift assays (EMSAs) have defined two fragments of the ALAS1 coding region that can bind to HepG2 cytosolic protein. However, this binding does not seem to be affected by the addition or depletion of haem. From the data presented in this thesis, a mechanism into how haem destabilises the ALAS1 mRNA in humans has been proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available