Incorporation of an internal ribosome entry site-dependent mechanism in arsenic-induced GADD45α expression

We have previously shown that trivalent arsenic (arsenite, As³⁺) is able to induce GADD45α expression in human bronchial epithelial cells through activation of c-Jun NH₂-terminal kinase and nucleolin-dependent mRNA stabilization. In the present report, we show that As³⁺ is capable of inducing translation of the GADD45α protein through a cap-independent, or rather, an internal ribosome entry site (IRES)-dependent mechanism. In growth-arrested cells, As³⁺ elevated the GADD45α protein level in a dose- and time-dependent manner which did not correlate with the GADD45α mRNA expression. Pretreatment of the cells with rapamycin, an inhibitor for the cap-dependent translation machinery through the suppression of mTOR and p70S6 kinase, failed to affect the induction of the GADD45α protein induced by As³⁺. Sequence analysis revealed a potential IRES element in the 5′-untranslated region of the GADD45α mRNA. This IRES element in the 5′-untranslated region of the GADD45α mRNA is functional in mediating As³⁺-induced translation of the GADD45α protein in a dicistronic reporter gene activity assay. Immunoprecipitation and proteomic studies suggest that As³⁺ impairs the assembly of the cap-dependent initiating complex for general protein translation but increases the association of human elongation factor 2 and human heterogeneous nuclear ribonucleoprotin with this complex. Thus, these results suggest that in growth-arrested cells, As³⁺ is still capable of inducing GADD45α expression through an IRES-dependent translational regulation.