An mRNA Loop/Bulge in the Ferritin Iron-responsive Element Forms in Vivo and Was Detected by Radical Probing with Cu-1,10-phenantholine and Iron Regulatory Protein Footprinting

Ke, Yaohuang; Theil, Elizabeth C.

doi:10.1074/jbc.c100614200

Cited by 23 publications

(29 citation statements)

References 26 publications

(41 reference statements)

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“…Several studies have shown that IRPs have different affinities for various IRE structures (34,35,48). IRP2 binds with greater affinity to the ferritin mRNA IRE than to the IREs of the TfR mRNA; this is attributed to the internal bulge found in the stem of the ferritin IRE (28,29). The crucial role of IRP2 in controlling ferritin levels in vivo also was indicated in recent studies demonstrating ferritin accumulation in the brains of IRP2 knockout mice (19).…”

Section: Discussionmentioning

confidence: 90%

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Mayo

Kohlhepp

Zhang

et al. 2004

American Journal of Physiology-Lung Cellular and Molecular Phys

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Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.

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Section: Discussionmentioning

confidence: 90%

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Mayo

Kohlhepp

Zhang

et al. 2004

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…The site of Cu-(phen) 2 binding in the helix of the ferritin mRNA is the same site associated with metal binding, proton-induced disorder, and IRP2 binding in solution (17,32). Recent studies with HeLa cells confirm that the specific RNA fold of the ferritin IL/B, recognized by Cu-(phen) 2 , occurs in vivo as well (56). In the present study, using the wild-type TfR-3Ј-UTR RNA (5xIRE), Cu-(phen) 2 mediated the cleavage of nucleotides in the C-bulge region of TfR-IREc (Fig.…”

Section: Mtementioning

confidence: 82%

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

Erlitzki

Long

Theil

2002

Journal of Biological Chemistry

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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.

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“…Chimeric RNAs between AMV and IRE, a proven PTL, were infectious to plants only when a transloop base pair at positions 1 and 5 of the hexanucleotide loop could form, indicating that the IRE PTL forms under in vivo circumstances. Recently, evidence for the structure of the ferritin IRE stem-loop in vivo was obtained by radical probing and protein footprinting in HeLa cells (26).…”

Section: Discussionmentioning

confidence: 99%

A plant virus replication system to assay the formation of RNA pseudotriloop motifs in RNA–protein interactions

Haasnoot

Bol

Olsthoorn

2003

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

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A pseudotriloop is formed by transloop base pairing between the first (5 ) and the fifth nucleotide in a hexanucleotide RNA loop (''hexaloop'') to subtend a triloop of nucleotides 2-4. This structure has been found in hairpins involved in the regulation of iron metabolism in mammalian cells and in transcription of plant virus subgenomic RNA. Several hexaloop hairpins, including HIV-transactivation-responsive element and hepatitis B virus , potentially adopt a pseudotriloop conformation. Here we show that an RNA plant virus whose replication depends on a conventional triloop hairpin can be used to verify the existence of pseudotriloop structures in vivo. Our data suggest that the pseudotriloop may represent a common motif in RNA-protein recognition.R NA-protein interactions play a fundamental role in various cellular processes and viral infections. Unlike DNA-binding proteins, which recognize sequence-specific motifs, proteins that interact with RNA often recognize structural features such as hairpin loops, bulges, and internal loops. The number of solved RNA-protein complexes has grown dramatically in recent years and has provided insight into the mechanisms of RNA recognition (1, 2). However, RNA motifs inferred from these studies are usually restricted to a specific class of related proteins and are not widely applicable.The family Bromoviridae comprises plant viruses with a tripartite RNA genome of messenger polarity (Fig. 1A). RNAs 1 and 2 encode the viral subunits of the RNA-dependent RNA polymerase (RdRp). The dicistronic RNA 3 encodes the movement protein (P3) and coat protein (CP). The latter is translated from a subgenomic (sg) mRNA 4. The best-studied members of this family are alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) (3, 4).For BMV we found that in vitro sgRNA synthesis relies on the presence of a small hairpin with a CAUAGA loop, but that a conventional trinucleotide ''triloop''-terminated hairpin could functionally replace this hexaloop-terminated hairpin structure (5). Using biochemical and biophysical techniques, we demonstrated that the CAUAGA loop adopts a triloop conformation by transloop base pairing of C1 and G5, creating a AUA triloop and bulging out A6. This conformation, which we have termed ''pseudotriloop'' (PTL), resembles the structure of iron-responsive elements (IREs) that regulate translation of mRNAs encoding proteins involved in iron uptake, storage, and utilization in mammalian cells. In the conserved loop motif CAGUGH (H ϭ A, C, or U) of IREs, a similar transloop base pair between C1 and G5 is essential for recognition by iron-regulatory proteins (6 -9). In contrast to the IRE loop, deletion of the bulge (A6) in the BMV loop was not detrimental for subgenomic promoter (sgp) activity. This finding suggested that the BMV RdRp is able to use both triloop and PTL hairpins for sg transcription (5).We demonstrated (10) that AMV RdRp also recognizes a triloop hairpin to direct sgRNA synthesis in vitro (Fig. 1B). Given its relatedness to BMV, we anticipated that AMV RdRp ...

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An mRNA Loop/Bulge in the Ferritin Iron-responsive Element Forms in Vivo and Was Detected by Radical Probing with Cu-1,10-phenantholine and Iron Regulatory Protein Footprinting

Cited by 23 publications

References 26 publications

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

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

A plant virus replication system to assay the formation of RNA pseudotriloop motifs in RNA–protein interactions

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