Iron Regulation and the Cell Cycle: IDENTIFICATION OF AN IRON-RESPONSIVE ELEMENT IN THE 3′-UNTRANSLATED REGION OF HUMAN CELL DIVISION CYCLE 14A mRNA BY A REFINED MICROARRAY-BASED SCREENING STRATEGY

Mayka Sanchez,Bruno Galy,Thomas Dandekar,Peter Bengert,Yevhen Vainshtein,Jens Stolte,Martina U Muckenthaler,Matthias W Hentze

doi:10.1074/jbc.m603876200

Abstract

Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5'- or 3'-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. IRP/IRE messenger ribonucleoproteins were immunoselected, and their mRNA composition was analyzed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Among different candidates, this report focuses on a novel IRE located in the 3'-untranslated region of the cell division cycle 14A mRNA. We show that this IRE motif efficiently binds both IRP1 and IRP2. Differential splicing of cell division cycle 14A produces IRE- and non-IRE-containing mRNA isoforms. Interestingly, only the expression of the IRE-containing mRNA isoforms is selectively increased by cellular iron deficiency. This work describes a new experimental strategy to explore the IRE/IRP regulatory network and uncovers a previously unrecognized regulatory link between iron metabolism and the cell cycle.

Highlights

Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5؅- or 3؅-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC)
To explore the IRE/IRP regulatory network, we immunopurified IRP1/mRNA complexes formed with RNA isolated from various cell lines and tissues and identified novel IRP binding mRNAs using the IronChip cDNA microarray platform [20]; here, the IronChip was complemented with genes bearing IRElike motifs that we identified by bioinformatic screens of nucleic acid databases
Embryonic lethality in mice deficient for both IRP1 and IRP2 shows that the IRP/IRE regulatory network is vital [9]

Summary

Iron Regulation and the Cell Cycle

IDENTIFICATION OF AN IRON-RESPONSIVE ELEMENT IN THE 3؅-UNTRANSLATED REGION OF HUMAN CELL DIVISION CYCLE 14A mRNA BY A REFINED MICROARRAY-BASED SCREENING STRATEGY*□S. To explore the IRE/IRP regulatory network, we immunopurified IRP1/mRNA complexes formed with RNA isolated from various cell lines and tissues and identified novel IRP binding mRNAs using the IronChip cDNA microarray platform [20]; here, the IronChip was complemented with genes bearing IRElike motifs that we identified by bioinformatic screens of nucleic acid databases. We demonstrate that this integrated experimental strategy reliably identifies known IRE-containing genes, and we report the identification of a conserved IRE in the 3Ј-UTR of mRNA encoding the CDC14A tumor suppressor gene, pointing to a previously unrecognized regulatory link between iron metabolism and the cell cycle

EXPERIMENTAL PROCEDURES

IRE at IRE sequence

IRE in other species

The other tested tissues and cell lines did not detectably express

Characterization of the Human

DISCUSSION