Identification of RNA-binding surfaces in iron regulatory protein-1

Kaldy, Pierre; Menotti, Eric; Moret, Rémy; Kühn, Lukas C.

doi:10.1093/emboj/18.21.6073

Cited by 48 publications

(52 citation statements)

References 58 publications

(106 reference statements)

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“…It is presumed to be structurally homologous to the mitochondrial aconitase (mAcn), which does not bind RNA (Robbins and Stout 1989). The binding site for RNA was modeled in the cleft of the two domains (Basilion et al 1994;Hirling et al 1994;Kaldy et al 1999). …”

Section: Introductionmentioning

confidence: 99%

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations

Hall¹,

Williams²

2003

RNA

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The iron responsive element (IRE) RNA hairpin loop contains six phylogenetically conserved nucleotides, which constitute part of the sequence-specific binding site of the IRE-binding protein. The NMR structure of the loop has been solved, showing that 3 of the 6 nt are poorly constrained. Here, two purine nucleotides in the IRE loop are individually replaced with the fluorescent purine analog 2-aminopurine (2AP). Steady-state and time-resolved fluorescence methods are used to describe the structure and dynamics of 2AP in the IRE loop. The data indicate that 2AP at the position of the adenosine in the loop moves between stacked and unstacked positions, whereas 2AP at the adjacent guanosine is predominantly solvent exposed. Stochastic dynamics simulations are used to provide a physical description of how those nucleotides might move.

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Section: Introductionmentioning

confidence: 99%

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations

Hall¹,

Williams²

2003

RNA

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“…S711 is located near R699, which is required for citrate͞isocitrate binding, and R728, R732, and other residues involved in RNA binding (15), suggesting that phosphorylation might target one or both of these functions. Interestingly, the S711 region is highly conserved in bacterial acnA enzymes and in IRP1͞c-acon in species from Drosophila to humans, whereas in IRP2, which is not an aconitase, this region also is well conserved with the exception that residue 711 is an Ala.…”

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confidence: 99%

Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Pitula

Deck

Clarke

et al. 2004

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

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Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the ironsulfur enzyme aconitase (c-acon). Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon. The role of PKC-dependent phosphorylation of S711 on IRP1͞c-acon function was examined. Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator. In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein. Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable. In contrast, when measured by the conversion of isocitrate to cis-aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function. This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios. Iron-responsive element RNA-binding affinity was unaffected by S711 mutations. Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate͞isocitrate metabolism. I t is critical that organisms balance the metabolic requirement for iron with its potential toxicity. Vertebrates possess an elegant system to sense cellular iron status, regulate the uptake and metabolic fate of iron, and thereby maintain iron homeostasis. Iron-regulatory protein 1 (IRP1) and IRP2 are sensory components of this critical regulatory network that promote increased iron uptake when cells are iron depleted and storage of iron when cells are iron overloaded (1, 2). IRPs control mRNA fate by binding to iron-responsive elements (IRE) in the untranslated regions of mRNA encoding proteins that control iron homeostasis. The RNA-binding activity of IRP can be regulated by multiple factors. Iron promotes the loss of IRP1 RNA-binding activity through insertion of a [4Fe-4S] cluster into the protein, converting it to the cytosolic isoform of the tricarboxylic acid (TCA) cycle enzyme aconitase (c-acon). Solvent accessibility of the Fe-S cluster in aconitases allows for perturbants to promote loss of the cluster from c-acon, converting it to IRP1 (1, 2). Consequently, reactive species such as NO and H 2 O 2 are able to promote removal of the Fe-S cluster, favoring generation of IRP1 from c-acon, although H 2 O 2 may do so through direct and indirect mechanisms (reviewed in refs. 1 and 2).Whereas much is known a...

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“…To create a B. subtilis strain expressing an enzymatically active but RNA binding-deficient aconitase, we based our approach on the mutagenesis of IRP-1 by Kaldy et al (23). Those authors concluded that arginines 728 and 732 in IRP-1 are critical for interaction of IRP-1 with RNA targets.…”

Section: Resultsmentioning

confidence: 99%

Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006

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Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of K -dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3 untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.Aconitase catalyzes the reversible isomerization of citrate and isocitrate in the second step of the tricarboxylic acid branch of the Krebs citric acid cycle. In Bacillus subtilis, the Krebs cycle is induced during late exponential phase in nutrient-exhausted medium and is responsible for the production of biosynthetic intermediates and ATP and for reducing power during stationary phase and sporulation. When induced, aconitase becomes one of the most abundant proteins in the cell (8). Induction of the aconitase gene, citB, is dependent on inactivation of two DNA binding proteins, CcpC (22) and CodY (25). Repression by CcpC is antagonized by citrate (22), whereas CodY loses repressing activity when the intracellular pools of GTP (32) and branched-chain amino acids (38) decrease. A citB null mutant is severely defective in spore formation and has a relative sporulation efficiency of 1.4 ϫ 10 Ϫ4 % compared to its wild-type parent (6). Previous work by Craig et al. (6) showed that the defect in sporulation of a citB null mutant is caused, in part, by the accumulation of citrate, a chelator of Mn 2ϩ and Fe 2ϩ . Chelation of divalent cations disrupts phosphorylation (activation) of Spo0A, the major transcription factor for early sporulation-specific genes (19). However, addition of excess Mn 2ϩ and Fe 2ϩ only partially overcomes the sporulation defect of an aconitase null mutant (6).When a constitutively active allele of spo0A is introduced into the citB null mutant, cells proceed past the stage 0 block in sporulation but are still blocked at a later stage (6). A similar result is seen when an aconitase null mutant is genetically modified to prevent synthesis of citrate (6); such cells proceed past stage 0 but do not complete sporulation efficiently. Taken together, these results suggest that citrate accumulation may be responsible for the stage 0 block, but aconitase may have an additional function unrelated to citrate metabolism that is important during the later stages of sporulation. The only other known activity of B. su...

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Identification of RNA-binding surfaces in iron regulatory protein-1

Cited by 48 publications

References 58 publications

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations

Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

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