Abstract
Iron regulatory factor (IRF) is a cytoplasmic RNA-binding protein involved in regulating iron homeostasis. IRF controls expression of ferritin and transferrin receptor post-transcriptionally via specific binding to stem-loop iron-responsive elements (IREs) located in the untranslated regions of the respective mRNAs. We have confirmed by RNA band-shift analysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulated by intracellular iron levels. This faster migrating complex appears to represent a specific interaction between the ferritin IRE and an iron-regulated protein that is distinct from IRF, as concluded from the following lines of evidence. First, UV cross-linking and partial digestion with different proteases revealed different peptide patterns for the two IRE-protein complexes. Second, antiserum raised against IRF peptides immunoprecipitated only authentic IRF and not the protein of the faster migrating complex, as determined by band-shift analysis. Following separation of the two IRE-binding proteins by ion-exchange chromatography, only the IRF-containing fraction reacted with the antibodies on Western blots. The second protein binds IREs with an affinity similar to that of IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. UV cross-linking experiments indicate that this second protein, tentatively named IRFB, has a molecular mass of approximately 105 kDa. Analysis of mouse tissues revealed differences in the distribution of IRF and IRFB. Whereas IRF protein and IRE binding activity were predominant in liver, intestine, and kidney, the IRFB protein(s) revealed highest binding activity in intestine and brain. Our data support the existence of two distinct iron-regulated IRE-binding proteins in rodents.
Highlights
Iron regulatory factor (IRF)is a cytoplasmic RNA- and theinverse effect under high iron, invoke post-transcripbinding proteininvolved in regulating ironhomeosta- tional mechanisms of gene regulation
By analogy to recent studies that indicate binding of multiple cellular factors to the TAT activation region (TAR) RNA stem-loop of HIV (for review, see Rosen (1991)), one might suppose that proteins other than IRF may bind nant in liver, intestine, and kidney, the IRFB proteinw(sit)h high affinity to the ferritin iron-responsive elements (IREs)
Previously in mouse Ltk- cells (Rothenberger et al, 1990)and The weaker intensity of the Ltk- complex B peptide pattern rat cell extracts (Leibold and Munro, 1988).To confirm these compared with that of IRF reflects the relative levels of these earlier observations, we performed experimentsusing two IRE-protein complexes detected by band-shift assay
Summary
Iron regulatory factor (IRF)is a cytoplasmic RNA- and theinverse effect under high iron, invoke post-transcripbinding proteininvolved in regulating ironhomeosta- tional mechanisms of gene regulation. The untranslated regionosf the respective mRNAs. We have confirmed by RNA band-shiftanalysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulatedby intracellular iron levels. We have confirmed by RNA band-shiftanalysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulatedby intracellular iron levels This faster migratingcomplex appears to representa specific interaction between the ferritin IRE and an iron-regulated proteitnhat isdistinct from (for review, see KlausnerandHarford (1989), Kuhnand Hentze (1992)). The second protein binds IREs with an affinity similar to thatof IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. U V cross-linkingexperimentsindicate thatthis second protein, tentativelynamed IRFB,has a molecular mass of -105 kDa. Analysis of mouse tissues revealed differences in the distributionof IRF andIRFB.Whereas IRF protein and IRE binding activity were predomiet al., 1991) mRNAs implicate IRF asa potentially important regulator of erythroid protoporphyrin biosynthesis and the citric acid cycle.
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