Abstract
Within the 5'-untranslated region of ferritin mRNAs, there is a conserved region of 28 nucleotides (nt) (the iron regulatory element (IRE)) that binds a protein (the IRE-binding protein (IRE-BP)) involved in the iron regulation of ferritin mRNA translation. We have examined the role of RNA secondary structure on the interaction of the IRE with the IRE-BP. First, the rat light ferritin IRE possesses a structure similar to that of the bullfrog heavy ferritin IRE (Wang, Y.-H., Sczekan, S. R., and Theil, E. C. (1990) Nucleic Acids Res. 18, 4463-4468). This includes an extended stem, interrupted at various points by bulge nucleotides and a 6-nt single-stranded loop (CAGUGU) at its top. Computer predictions and mapping results suggest the presence of a 3-nt (UGC) bulge 5 bases 5' of the loop in the rat IRE. Second, disruption of the base pairing in the upper stem alters IRE secondary structure and reduces the affinity with which the IRE-BP binds the IRE. Third, increasing the size of the loop or the distance between the UGC bulge and the loop reduces the IRE/IRE-BP interaction. Our results indicate that several aspects of IRE secondary structure are important for its high affinity binding to the IRE-BP.
Highlights
From the YUnited States Departmentof Agriculture, Human Nutrition Research Center onAging, Tufts University, Boston, Massachusetts 02111 and the $Division of Toxicology, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139
(nt) (the iron regulatory element (IRE)) that binds a UTR of an mRNA provides for iron-dependent regulation of protein (the IRE-binding protein (IRE-BP)) involved mRNA translation, whereas if present in the 3’-UTR, toin the iron regulation of ferritin mRNA translation. getherwith other RNA sequences, IREs can provide iron
Both computer modeling and nuclease mapping experistem, interrupted at various points by bulge nucleoments have indicated that the IRE forms the central part of tides and a 6-nt single-strandedloop (CAGUGU)at its a large stem-and-loop structure near the 5’-end of ferritin top
Summary
’Wisconsin, 1415 Linden Dr, Madison, WI 53706. The abbreviations used are: L- and H-ferritin, light and heavy ferritin, respectively; IRE, iron regulatory element; IRE-BP, IREbinding protein; nt, nucleotide; TfR, transferrin receptor; UTR, untranslated region. TfR IREsequences [10,11], the sixthnucleotide of the loop may apparently be an adenosine. The preferred nucleotide is a pyrimidine in 90% of the ferritin IREs and in 80% of the TfR IRES for which sequence information is available. EXPERIMENTALPROCEDURES4 mutant RS, combination of mutants 5‘M and 3“ to restore pairingin the topmoststem, but with a differentprimary
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