Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress

Abstract

Cellular inhibitor of apoptosis protein 1 (c-IAP1) can regulate apoptosis through its interaction with downstream TNF receptor effectors (TRAF1 and TRAF2), by binding to and inhibiting certain caspases, and by controlling the levels of specific proapoptotic stimuli (e.g., Smac/DIABLO) within the cell. Studies involving the expression of c-IAP1 mRNA and protein in cells and tissues have provided evidence suggesting c-IAP1 expression may be posttranscriptionally controlled. Because the 5′-UTR of c-IAP1 mRNA is unusually long, contains multiple upstream AUG codons, and has the potential to form thermodynamically stable secondary structures, we investigated the possibility it contained an internal ribosome entry site (IRES) that may regulate its expression. In the present study, the c-IAP1 5′-UTR exhibited IRES activity when dicistronic RNA constructs were translated in rabbit reticulocyte lysate (RRL) and in transiently transfected cells. IRES-mediated translation was similar to that exhibited by the hepatitis C virus IRES but varied significantly in RRL and in HeLa, HepG2, and 293T cells, indicating the c-IAP1 IRES was system and cell type specific. IRES-mediated translation was maintained in mono- and dicistronic constructs in which the UTR was inserted downstream from a stable hairpin that prevented cap-dependent ribosome scanning. In cells, the presence or absence of a methylated cap did not significantly affect the translation of polyadenylated, monocistronic RNAs containing the c-IAP1 5′-UTR. IRES-mediated translation was stimulated in transfected cells treated with low doses of pro-apoptotic stimuli (i.e., etoposide and sodium arsenite) that inhibited endogenous cellular translation.

Keywords

• translation
• apoptosis
• internal ribosome entry site
• inhibitor of apoptosis protein
• IAP, inhibitor of apoptosis protein
• eGFP, enhanced green fluorescence protein
• β-Gal, beta-Galactosidase
• ORF, open reading frame
• UTR, untranslated region
• uAUG, upstream AUG codon
• iAUG, initiator AUG codon
• RNP, ribonucleoprotein
• SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis