The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution

Abstract

A post-transcriptional regulatory protein, termed iron regulatory factor (IRF), that binds specifically to the iron-responsive elements of ferritin and transferrin receptor mRNA, has recently been identified in the cytoplasm of human and mouse cells. Activation of this factor by low intracellular iron levels leads to inhibition of ferritin translation and an increase of TR mRNA stability. To investigate whether these feedback regulatory mechanisms are conserved during evolution, we analysed cytoplasmic extracts from 12 different species for a specific IRE-binding activity. We found mRNA-binding proteins in chicken, frog, fish and fly, which are equivalent to human and mouse IRF in gel-retardation assays with radiolabeled RNA transcripts. Competition experiments, molecular weight determinations, and modulation of the mRNA-binding activity in response to intracellular iron levels or reduction by beta-mercaptoethanol indicate that IRF has similar structural and functional properties in these different species.

Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase.

“…Upon modulation of iron levels, IRE binding of hIRF was apparently induced as for the endogenous mouse IRF. A difficulty arose, however, in the interpretation of these data, since mouse extracts contain a second IRE-binding protein (mIRFB; Henderson et al, 1993) which forms an RNA-protein complex with H-chain ferritin IRE that co-migrates with the complex of human IRF ( Figure 7A; Rothenberger et al, 1990). Recently, we have found a solution to this problem by selecting an IRE variant that binds well to IRF, but does not recognize mIRFB.…”

“…Where indicated, radiolabeled IRE probe was added together with 2-mercaptoethanol. The incubations and separation of IRE-IRF complexes from unbound probe were carried out by non-denaturing gel electrophoresis as previously described (Rothenberger et al, 1990). In some experiments, IRF was preincubated with either 1.5 mM azodicarboxylic acid bis [dimethylamide] (Sigma, St Louis, MO), known as diamide, or different concentrations of N-ethylmaleimide, or 5 mM iodoacetamide, or 10 mM K3Fe(CN)6 with 0.1 mM EDTA, or 2% 2-mercaptoethanol, or various concentrations of cis-aconitate as mentioned in the results.…”

Ferritin mRNAs in Schistosoma Mansoni do not have Iron‐Responsive Elements for Post‐Transcriptional Regulation

“…After a 30-min incubation on ice, the extracts were centrifugated at 4°C for 10 min at 12000 g. Cell-free supernatants were used for RNA binding experiments as described by Rothenberger et al (1990).…”

“…The IRE is found in all fully characterized ferritin mRNAs of higher animals (Theil, 1994), with only one exception, the socalled yolk ferritin of Lyriinnea (von Darl et al, 1994). In contrast, ferritins of plants, yeast and bacteria do not possess the IRE element (Lobreaux et al, 1993;Rothenberger et al, 1990;Theil, 1987). The IRE is a highly conserved sequence/structure RNA motif forming a distinct stem loop which is located in close proximity to the cap site of ferritin mRNAs (Goossen et al, 1990).…”

Altered iron homeostasis involvement in arsenite-mediated cell transformation

“…Kuühn, ISREC, Switzerland), which contains human ferritin H chain IRE. RNA gel-shift assay was performed as previously described [43]. Briefly, samples were equally divided into two parts; 2-mercaptoethanol (2-ME) was added to one part of the sample at a 2% final concentration.…”