Internal Loop/Bulge and Hairpin Loop of the Iron-Responsive Element of Ferritin mRNA Contribute to Maximal Iron Regulatory Protein 2 Binding and Translational Regulation in the Iso-iron-responsive Element/Iso-iron Regulatory Protein Family

Ke, Yaohuang; Sierzputowska-Gracz, Hanna; Gdaniec, Zofia; Theil, Elizabeth C.

doi:10.1021/bi9924765

Cited by 51 publications

(69 citation statements)

References 46 publications

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“…1), which differs from human ferritin-L and frog ferritin IRE only by having a C-G closing base pair rather than a U-G, the G 26 residue, also in the internal loop region of the IRE, was not protected by yohimbine from oxidation. This result is consistent with the observation that G 26 and G 27 exhibit different environments when probed by NMR and metal-binding studies (43). The native fluorescence of yohimbine was also used to study binding to the IRE.…”

Section: Resultssupporting

confidence: 88%

“…In contrast, addition of Hoechst 33258, which is well known to bind nucleic acids (49), inhibited ferritin synthesis by Ͼ50% (data not shown). As shown in this (see supporting information) and earlier work (16,43), a G 18 A mutation in the ferritin IRE (G 18 forms a base pair with C 14 in the IRE terminal loop) (33, 34) displays a reduced affinity for IRP (16,43), which increases ferritin mRNA translation. Translation of mRNA with the G 18 A mutation in the IRE was unaffected by yohimbine (Fig.…”

Section: Acceleration Of Ferritin Synthesis By Yohimbinesupporting

confidence: 65%

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The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

Tibodeau

Fox²,

Ropp³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE-yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA.footprinting ͉ protein synthesis ͉ RNA structure ͉ RNA-binding drugs ͉ gene expression T he targeting of small molecules to three-dimensional structures in RNA has the potential to change the synthesis rate of the encoded proteins (1-4); an example is alteration of the control of reverse-transcription rate rates of HIV-1 RNA by trans-activation responsive region RNA, a target of many druglike molecules (5-9). A principal challenge in targeting mRNA is identifying three-dimensional RNA structures that are functionally relevant and exhibit structures amenable to selective binding in the presence of large quantities of other nucleic acids (5-7). Recent findings on the activation of bacterial riboswitches by metabolites provide incentive to pursue mRNA binding as a new avenue for pharmaceutical research (10), particularly given the intriguing possibility that such elements exist in eukaryotes (11,12). The advantages of using drug-like molecules to target three-dimensional RNA targets include the ability to escape biological systems that respond to targeting helical RNA structures (13), the ability to identify potentially useful RNA targets in genome sequences (14,15), and the possibility of synthesizing reasonably large quantities of RNA for screening by chemical methods (2,16). An additional appeal is that chemicalfootprinting techniques, which are currently more ...

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Section: Resultssupporting

confidence: 88%

Section: Acceleration Of Ferritin Synthesis By Yohimbinesupporting

confidence: 65%

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

Tibodeau

Fox²,

Ropp³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Third, genetic ablation of both IRPs in intestine dramatically increased ferritin and ferroportin protein levels yet only modestly increased m-acon protein, apparently because ferritin and ferroportin mRNA translation are more strongly repressed by IRP action . These findings, along with the observation that IRPs appear to differentially interact with the ferritin and m-acon IREs, suggest that IREs differ in their regulatory potential for dictating control of mRNA translation Ke et al 2000). In this study, we focused on determining if an RNA binding hierarchy exists between IRP1 and 59 IREs and what sequences within these regulatory elements modulate this hierarchy.…”

Section: Introductionmentioning

confidence: 99%

Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy

Goforth¹,

Anderson²,

Nizzi³

et al. 2009

RNA

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Iron regulatory proteins (IRPs) are iron-regulated RNA binding proteins that, along with iron-responsive elements (IREs), control the translation of a diverse set of mRNA with 59 IRE. Dysregulation of IRP action causes disease with etiology that may reflect differential control of IRE-containing mRNA. IREs are defined by a conserved stem-loop structure including a midstem bulge at C8 and a terminal CAGUGH sequence that forms an AGU pseudo-triloop and N19 bulge. C8 and the pseudo-triloop nucleotides make the majority of the 22 identified bonds with IRP1. We show that IRP1 binds 59 IREs in a hierarchy extending over a ninefold range of affinities that encompasses changes in IRE binding affinity observed with human L-ferritin IRE mutants. The limits of this IRE binding hierarchy are predicted to arise due to small differences in binding energy (e.g., equivalent to one H-bond). We demonstrate that multiple regions of the IRE stem not predicted to contact IRP1 help establish the binding hierarchy with the sequence and structure of the C8 region displaying a major role. In contrast, base-pairing and stacking in the upper stem region proximal to the terminal loop had a minor role. Unexpectedly, an N20 bulge compensated for the lack of an N19 bulge, suggesting the existence of novel IREs. Taken together, we suggest that a regulatory binding hierarchy is established through the impact of the IRE stem on the strength, not the number, of bonds between C8 or pseudo-triloop nucleotides and IRP1 or through their impact on an induced fit mechanism of binding.

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“…Recently, metal-sensitive quadruplexes have been shown to function as a protein synthesis rate regulator in a human mRNA (ADAM10;19,20). Our earlier studies showed direct binding of a number of nonferrous metal ions to IRE-RNA, such as Mg 2þ (21,22) and Mn 2þ (12) as well as complexes with Cu 1þ (21) and Ru 2þ (23).Regulation of stable mRNAs permits rapid cellular responses. Eukaryotic protein synthesis can be divided into three main phases: initiation, elongation, and termination.…”

mentioning

confidence: 99%

“…A large RNA surface remains exposed, inviting RNA interactions with other molecules and ions even while bound to IRP1. Probing IRE-mRNA with chemical cleavage agents such as Cu 1þ -1,10-phenanthroline or Mg 2þ (21,22) to locate metalsensitive sites, combined with the X-ray crystal structure of the IRE-RNA/IRP1 complex, shows that some metal sites are located on exposed RNA surfaces and are available for additional RNA interactions even in the IRP1 complex ( Fig. 1; 28).…”

mentioning

confidence: 99%

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

Haldar²,

Khan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe 2þ . The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe 2þ (anaerobic) or Mn 2þ selectively increase the IRE-RNA/IRP K D . Here we show: (i) Fe 2þ binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe 2þ increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.ferrous ion regulation | metabolic riboregulator I ron increases rates of ferritin protein synthesis in animals by facilitating messenger RNA/ribosome binding; metabolic iron (i.e., "labile" or "free" iron in cells) is considered to be ferrous (1). The iron response requires a noncoding riboregulator called the "iron-responsive element" (IRE), which is approximately 30 nt, folded into a distorted, bulged helix loop (2-5). This riboregulatory structure is also found in mRNAs for proteins of iron traffic (6-9), cell cycling (10), and the nervous system (11). IRP proteins bind with different stabilities to IRE-RNAs of the IRE-RNA family (12, 13), creating a graded or hierarchal set of mRNA responses to iron in vivo. Deletion of the 30 nt IRE-RNA not only removes IRP regulation but also decreases the rate of IRP-independent protein synthesis (14). A number of current models of IRE-RNA/IRP regulation feature iron-dependent degradation/modification of the IRP proteins as the main control point (8,9,15,16). Such models do not answer two important questions: (i) How does iron increase release of IRP protein for [4Fe-4S]-modification and/or degradation? Overlap of the IRE-RNA and the Fe-S binding sites on IRP1 prevents Fe-S insertion in the IRP1/IRE-RNA complex (5). (ii) How does the IRE-RNA control rates of IRP-independent protein synthesis (14)? In an earlier study, we showed that Fe 2þ ions (anaerobic) selectively increased the dissociation constant for the IRE-RNA/IRP1 complex in solution (12). Here we r...

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Internal Loop/Bulge and Hairpin Loop of the Iron-Responsive Element of Ferritin mRNA Contribute to Maximal Iron Regulatory Protein 2 Binding and Translational Regulation in the Iso-iron-responsive Element/Iso-iron Regulatory Protein Family

Cited by 51 publications

References 46 publications

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy

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

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Internal Loop/Bulge and Hairpin Loop of the Iron-Responsive Element of Ferritin mRNA Contribute to Maximal Iron Regulatory Protein 2 Binding and Translational Regulation in the Iso-iron-responsive Element/Iso-iron Regulatory Protein Family

Cited by 51 publications

References 46 publications

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy

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

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