In vivo generation of 3' and 5' truncated species in the process of c-myc mRNA decay

It has been demonstrated that the half-life of c-myc mRNA is modulated in response to physiological agents, The elucidation of the decay process and the identification of the critical steps in the in vivo c-myc mRNA degradation pathway can be approached by following the fate of c-myc mRNA under the influence of such factors, IFN-alpha was the factor used to modulate c-myc mRNA half-life in HeLa 1C5 cells, a stable clone derived from HeLa cells, This cell line carries multiple copies of the c-myc gene, under the control of the dexamethasone inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Exposure of HeLa 1C5 cells to IFN-alpha resulted in a further 2-fold increase over the dexamethasone-induced c-mye mRNA, However, the c-mye mRNA in IFN-alpha treated cells was less stable than that in the control cells. RNase H mapping of the 3' untranslated region of c-mye mRNA revealed, in addition to the full length mRNA, three smaller fragments, These fragments were proven to be truncated, non-adenylated c-mye mRNA species generated in vivo. Exposure of HeLa 1C5 cells to Interferon-alpha before induction with dexamethasone resulted in the enhanced presence of these intermediates. RNase H analysis of c-mye mRNA after actinomycin D chase revealed that deadenylation led to the formation of a relatively more stable oligoadenylated c-mye mRNA population which did not appear to be precursor to the truncated intermediates, The detection of truncated 3' end c-mye mRNA adenylated fragments as well, implies that the c-mye mRNA degradation process may follow an alternative pathway possibly involving endonucleolytic cleavage.