Identification of secondary structure in the 5′-untranslated region of the human adrenomedullin mRNA with implications for the regulation of mRNA translation

Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5′-untranslated region (5′ UTR) of human AM mRNA. Reverse transcriptase–polymerase chain reaction of the 5′ UTR consistently resulted, in addition to the product with the expected size of 155 base pair (bp), in a second product with an ∼65-bp deletion from the central region of the 5′ UTR, suggesting the presence of a secondary structure. The presence of a stem–loop structure was confirmed by probing the 5′ UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem–loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 5′ UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem–loop structure localized between nt +31 and +95 from the cap site led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 5′ UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 5′ UTR but lacking the region of secondary structure. Although we conclude that the 5′ UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem–loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.