AN ALTERNATIVELY-SPLICED 5'UNTRANSLATED EXON IN HUMAN ALAS1 INHIBITS TRANSLATION AND HAEM-REGULATED mRNA DESTABILISATION

Heme controls its own supply in non erythroid cells by regulating the stability of the mRNA encoding  5-amino-levulinate synthase (ALAS1), the first enzyme of the heme biosynthetic pathway. The human ALAS1 gene contains two non coding exons (1A and 1B) that are alternatively spliced to generate two mRNA species: a major one in which exon 1B is omitted and a ubiquitously expressed minor one, representing approximately 10% of the steady state concentration of ALAS1 mRNA, which contains exons 1A and 1B. We have investigated the role of this spliced form in regulated ALAS1 expression. Firstly, heme minimally affects the concentration of the minor mRNA species at a concentration that destabilises the major form. In addition, the 5' UTR of the minor mRNA inhibits, 8 fold, the translation of a heterologous reporter gene compared to the 5' UTR of the major mRNA species in HepG2 cells. These data suggest that the mechanism of heme-dependent destabilisation of ALAS1 mRNA may require its active translation. We have also investigated the sequence elements within of the minor mRNA that inhibit translation. Notably the 5' UTR contains 4 short overlapping upstream open reading frames (uORFS; U1-U4) that may inhibit cap-mediated translation by regulating the scanning of ribosomes to the ALAS1 ORF. Alternatively, the 5' UTR may contain an Internal Ribosomal Entry Site (IRES) that allows translation under stress conditions in which cap-mediated translation is compromised.