HAEM-MEDIATED DESTABILISATION OF HUMAN ALAS1 mRNA

Sadie J. Redding, AG Roberts and D Llewellyn

Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK

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 aminolaevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme in the haem biosynthetic pathway. However, the underlying mechanism is unknown and consequently our investigations have set out to determine exactly how haem regulates ALAS1 mRNA stability to control its own synthesis in non-erythroid cells.

The experimental approach to delineate the ALAS1 cis-acting element(s) has involved RT-PCRs and heterologous reporter gene assays to analyse the stability of ALAS1-fusion mRNAs in response to haem in the human HepG2 hepatoma cells. The data demonstrates that the sequences that confer haem-mediated instability reside within the ALAS1 mRNA coding region and translation through the coding region is required for haem-mediated destabilisation. A minor alternatively-spliced ALAS1 mRNA species is poorly translated and consequently resistant to haem-mediated decay. These results provide a basis for identifying both the cis- and trans-acting factors, and their interactions involved in haem-mediated instability of ALAS1 mRNA.