Ferritin L and H Subunits Are Differentially Regulated on a Post-transcriptional Level

Mimi C Sammarco,Scott Ditch,Ayan Banerjee,Ed Grabczyk

doi:10.1074/jbc.m703456200

Abstract

Ferritin plays an important role in the storage and release of iron, an element utilized in cellular processes such as respiration, gene regulation, and DNA replication and repair. Ferritin in animals is composed of 24 ferritin L (FTL) and ferritin H (FTH) subunits in ratios that vary in different cell types. Because the subunits are not functionally interchangeable, both L and H units are critical for maintaining iron homeostasis and protecting against iron overload. FTL and FTH are regulated primarily at a post-transcriptional level in response to cellular iron concentrations. Individual regulation of FTL and FTH is of much interest, and although transcriptional differences between FTL and FTH have been shown, differences in their post-transcriptional regulation have not been evaluated. We report here that FTL and FTH are differentially regulated in 1% oxygen on a post-transcriptional level. We have designed a quantitative assay system sensitive enough to detect differences between FTL and FTH iron regulatory elements (IREs) that a standard electrophoretic mobility shift assay does not. The FTL IRE is the primary responder in the presence of an iron donor in hypoxic conditions, and this response is reflected in endogenous FTL protein levels. These results provide evidence that FTL and FTH subunits respond independently to cellular iron concentrations and underscore the importance of evaluating FTL and FTH IREs separately.

Highlights

Ferritin is composed of L and H subunits that, highly conserved, are genetically separate [2, 3] and maintain distinct functions
Our study provides the first evidence that Ferritin L (FTL) and Ferritin H (FTH) are differentially regulated on a translational level
We confirmed the reliability of the construct and used this reporter system to demonstrate a measurable difference between IRP binding to FTL and FTH iron regulatory elements (IREs) in both normoxic and hypoxic conditions

Summary

Differential Regulation of Ferritin Subunits

IREs have only 49.5% identity, and because small changes in the nucleotide sequence in both stem and loop translate to larger changes in IRP binding ability (28 –31), differences in IRE structures may be the basis for differential translational regulation of L and H subunits. The result is that IRP1 and IRP2 bind overlapping but distinct sets of IRE targets [37]; this, combined with sequence variation in the known IREs, suggests a physiological graded response to iron To address this issue we designed a novel in vivo reporter system that isolates and quantifies IRE/IRP interaction, improving upon prior systems (38 – 40), with the advantages of co-expression from a bi-directional promoter in stably transfected cells. We exploited this reporter system to discern distinct differences in FTL and FTH translation in response to iron replete and iron deplete conditions in normoxic and hypoxic environments. This is the first study to quantify differential regulation of FTL and FTH at the translational level

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION