The mRNA-binding Site of Annexin A2 Resides in Helices C–D of its Domain IV

Annexin A2 (AnxA2) is a Ca2+-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific ∼ 100 nucleotide region in the 3′-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3′-untranslated region of anxA2 mRNA is Ca2+-dependent. By contrast, the interaction is Ca2+-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca2+-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.