Translational regulation of ferritin synthesis in rat liver. Effects of chronic dietary iron overload.

In rats with chronic dietary iron overload, a higher amount of liver ferritin L-subunit mRNA was found mainly engaged on polysomes, whereas in control rats ferritin L-subunit mRNA molecules were largely stored in ribonucleoprotein particles. On the other hand, ferritin H-subunit mRNA was unchanged by chronic iron load and remained in the inactive cytoplasmic pool. In agreement with previous reports, in rats acutely treated with parenteral iron, only the ferritin L-subunit mRNA increased in amount, whereas both ferritin subunit mRNAs shifted to polysomes. This may indicate that, whereas in acute iron overload the hepatocyte operates a translation shift of both ferritin mRNAs to confront rapidly the abrupt entry of iron into the cell, during chronic iron overload it responds to the slow iron influx by translating a greater amount of L-subunit mRNA to synthesize isoferritins more suitable for long-term iron storage.     

Both subunits of rat liver ferritin are regulated at a translational level by iron induction.

A few hours after administering iron to rats, liver ferritin synthesis increases several fold. However, Northern blot analysis with cDNA probes for ferritin light (L) and heavy (H) subunit mRNAs failed to show an increase in total population of either messenger. Cytoplasmic distribution of ferritin messages was therefore investigated in control and iron administered rats killed at 3.5 hours. The liver post-mitochondrial supernatant was fractionated on a sucrose gradient to separate polyribosomes, monosomes, ribosomal subunits and cell sap. RNA extracted from each fraction and analyzed using Northern blotting showed that 65% of the total mRNA population for each subunit was present in the cell sap of control rats, presumably as mRNP particles since ribosomal RNA was absent from this fraction. After iron administration, these reserves of free mRNA were recruited onto the polysomes, reducing the free mRNA pool to 15% of the total. We interpret this to be due to activation of blocked ferritin messages on entry of iron into the cell.     

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.     

Translational control of gene expression in a normal fibroblast. Characterization of a subclass of mRNAs with unusual kinetic properties

The translation of a small number of mRNAs in mouse SC-1 fibroblasts can be stimulated by cycloheximide, under conditions where the synthesis of most proteins is inhibited. These mRNAs are ordinarily present in small polyribosomes or messenger ribonucleoprotein particles, although the addition of cycloheximide drives them into large (greater than or equal to 5) polysomes. These mRNAs cannot be translated in vitro unless they are extracted with phenol. With such treatment, however, they are translated with normal competitive efficiencies. In iron-poor media, the mRNA for ferritin exhibits several of the distinctive kinetic properties of this class of mRNAs. With iron supplementation, however, ferritin translation appears normal. These observations are consistent with the existence of translational induction/repression systems in eukaryotes. Several types of evidence suggest that repressors may act by interfering with the interaction between mRNAs and limiting translational initiation components.     

Differential translational regulation of IRE-containing mRNAs in Drosophila melanogaster by endogenous IRP and a constitutive human IRP1 mutant

Insects, like vertebrates, express iron regulatory proteins (IRPs) that may regulate proteins in cellular iron storage and energy metabolism. Two mRNAs, an unspliced form of ferritin H mRNA and succinate dehydrogenase subunit b (SDHb) mRNA, are known to comprise an iron responsive element (IRE) in their 5'-untranslated region making them susceptible to translational repression by IRPs at low iron levels. We have investigated the effect of wild-type human IRP1 (hIRP1) and the constitutively active mutant hIRP1-S437 in transgenic Drosophila melanogaster. Endogenous Drosophila IRE-binding activity was readily detected in gel retardation assays. However, translational repression assessed by polysome gradients was only visible for unspliced IRE-containing ferritin H mRNA, but not for SDHb mRNA. Upon expression of exogenous hIRP1-S437 both mRNAs were strongly repressed. This correlated with a diminished survival rate of adult flies with hIRP1 and complete lethality with hIRP1-S437. We conclude that constitutive IRP1 expression is deleterious to fly survival, probably due to the essential function of SDHb or proteins encoded by yet unidentified target mRNAs.