Cleavage of Functionally Relevant Sites in Ferritin mRNA by Oxidizing Metal Complexes

IREs, a family of mRNA regulatory structures, control mRNA function and the synthesis of ferritin and the transferrin receptor. IRE secondary structure is a hairpin; in ferritin mRNA, the IRE also interacts with a base-paired flanking region (FL). Using Rh(phen) 2 phi 3+ and Ru(tpy)(bpy)O 2+ as cleavage reagents (phen = 1,10-phenanthroline, phi = 9,10-phenanthrenequinone diimine, tpy = 2,2‘,2‘‘-terpyridine, bpy = 2,2‘-bipyridine), two different substructures in the IRE or FL were detected that are related to regulation. Rh(phen) 2 phi 3+, which intercalates in RNA or DNA, exhibited a single major cleavage site in the FL. Mutation FL2 altered negative IRE regulation and eliminated the specific Rh(phen) 2 phi 3+ cleavage site; the FL2 derivative, FL2R, restored regulation and the Rh(phen) 2 phi 3+ site. Ru(tpy)(bpy)O 2+, which interacts with nucleic acids electrostatically and cleaves on the basis of solvent accessibility and chemical reactivity, cleaved a single site, G14, in the IRE hairpin loop. G14 appeared to have a simple structure with classical probes but, on the basis of Ru(tpy)(bpy)O 2+ cleavage, appears to be in a region of high accessibility, possibly caused by distortion in the phosphate backbone. Mutation IL-2 created a heptaloop and destroyed the Ru(tpy)(bpy)O 2+ site; positive and negative regulations were also affected in IL-2. IRE mutation at other sites did not affect the Ru(tpy)(bpy)O 2+ /RNA interaction. In addition to identifying substructure in the IRE+FL, Rh(phen) 2 phi 3+ and Ru(tpy)(bpy)O 2+ should also be useful in determining substructure of potential functional significance in other mRNAs as well as ribozymes, rRNAs, viral RNAs, and snRNAs.