Multiple, Conserved Iron-responsive Elements in the 3′-Untranslated Region of Transferrin Receptor mRNA Enhance Binding of Iron Regulatory Protein 2

Abstract

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3-untranslated region (TfR-3-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen) 2 binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.

Fe 2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation

“…Isolation of recombinant rabbit IRP1 from yeast used methods previously described in (12,42). The eIF4F protein was purified from rabbit reticulocyte lysate (RRL) as described previously (43).…”

Post‐transcriptional regulation of gene expression by degradation of messenger RNAs

“…This domain can form a hairpin structure in the human as well as rat VEGF 3 0 UTRs and presents 93% homology between the two species (Pages et al, 2000). The potential for recognition by RNA-binding proteins of stem-loop protein has been established in other systems (Richardson et al, 1998;Erlitzki et al, 2002). The presence of stem-loops in UTRs controlling mRNA translation, degradation, and localization has recently been reviewed .…”

Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

“…Bach1/DNA binding used the method described by Carey for protein-DNA interactions (32) and used by us for protein-RNA interactions (15,33,34 (36). The data (Table 1) are presented as the mean (n ϭ 3) with the error as the standard deviation.…”

“…The translational mechanism uses IRP1 and IRP2 to coordinate fth and ftl mRNA regulation with that of several other mRNAs important in iron and oxygen homeostasis by binding to iron-responsive elements (IRE) in each of the mRNAs. The IRE is a specific three-dimensional loop-helix-loop-helix structure (10) in the noncoding regions of the mRNAs (11)(12)(13)(14)(15)(16). Specific interactions between the iron regulatory proteins IRP1 and IRP2 and the different IREs in the mRNAs create a natural, combinatorial array of RNA⅐protein complexes (17).…”