CLEAVAGE OF FUNCTIONALLY RELEVANT SITES IN FERRITIN MESSENGER-RNA BY OXIDIZING METAL-COMPLEXES

IREs, a family of mRNA regulatory structures, control mRNA function an d the synthesis of ferritin and the transferrin receptor. IRE secondar y structure is a hairpin; in ferritin mRNA, the IRE also interacts wit h a base-paired flanking region (FL). Using Rh(phen)(2)phi(3+) and Ru( tpy)(bpyO2+ 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 detec ted that are related to regulation. Rh(phen)(2)phi(3+), which intercal ates in RNA or DNA, exhibited a single major cleavage site in the FL. Mutation FL2 altered negative IRE regulation and eliminated the specif ic Rh(phen)(2)phi(3+) cleavage site; the FL2 derivative, FL2R, restore d regulation and the Rh(phen)(2)phi(3+) site. Ru(tpy)(bpyO2+, which in teracts with nucleic acids electrostatically and cleaves on the basis of solvent accessibility and chemical reactivity, cleaved a single sit e, G14, in the IRE hairpin loop. G14 appeared to have a simple structu re with classical probes but, on the basis of Ru(tpy)(bpyO2+ cleavage, appears to be in a region of high accessibility, possibly caused by d istortion in the phosphate backbone. Mutation IL-2 created a heptaloop and destroyed the Ru(tpy)(bpyO2+ site; positive and negative regulati ons were also affected in IL-2. IRE mutation at other sites did not af fect the Ru(tpy)(bpyO2+/RNA interaction. In addition to identifying su bstructure in the IRE+FL, Rh(phen)(2)phi(3+) and Ru(tpy)(bpyO2+ should also be useful in determining substructure of potential functional si gnificance in other mRNAs as well as ribozymes, rRNAs, viral RNAs, and snRNAs.