Iron Regulatory Proteins 1 and 2 Bind Distinct Sets of RNA Target Sequences

Abstract

Iron regulatory proteins (IRPs) 1 and 2 bind with equally high affinity to iron-responsive element (IRE) RNA stem-loops located in mRNA untranslated regions and, thereby, post-transcriptionally regulate several genes of iron metabolism. In this study we define the RNA-binding specificities of mouse IRP-1 and IRP-2. By screening loop mutations of the ferritin H-chain IRE, we show that both IRPs bind well to a large number of IRE-like sequences. More significantly, each IRP was found to recognize a unique subset of IRE-like targets. These IRP-specific groups of IREs are distinct from one another and are characterized by changes in certain paired (IRP-1) or unpaired (IRP-2) loop nucleotides. We further demonstrate the application of such sequences as unique probes to detect and distinguish IRP-1 from IRP-2 in human cells, and observe that the IRPs are regulated similarly by iron and reducing agents in human and rodent cells. Importantly, the ability of each IRP to recognize an exclusive subset of IREs was conserved between species. These findings suggest that IRP-1 and IRP-2 may each regulate unique mRNA targets in vivo, possibly extending their function beyond the regulation of intracellular iron homeostasis.The regulation of cellular iron homeostasis is under the post-transcriptional control of iron regulatory protein-1 (IRP-1), 1 a cytoplasmic RNA-binding protein with specificity for mRNA stem-loop structures known as iron-responsive elements (IREs) (reviewed by Klausner et al. (1993) and Kü hn (1994)). IRP-1, formerly referred to as IRE-binding protein Leibold and Munro, 1988), iron regulatory factor (IRF; Mü llner et al., 1989), or ferritin repressor protein (Walden et al., 1988), has been identified as the cytosolic counterpart of the citric acid cycle enzyme, aconitase (Hentze and Argos, 1991;Rouault et al., 1991;Kaptain et al., 1991;Haile et al., 1992a;Kennedy et al., 1992). IRP-1 is now regarded as a bi-functional "sensor" of iron, switching between RNA binding and enzymatic activities depending on cellular iron status (Haile et al., 1992a(Haile et al., , 1992bConstable et al., 1992; EmeryGoodman et al., 1993;Basilion et al., 1994). In iron-depleted cells, IRP-1 inhibits translation of ferritin and erythroid 5-aminolevulinic acid synthase mRNAs by binding to IREs located in their 5Ј-untranslated region (UTR) (Aziz and Munro, 1987;Hentze et al., 1987;Bhasker et al., 1993;Melefors et al., 1993). Binding of IRP-1 to a cluster of five IREs in the 3Ј-UTR of transferrin receptor mRNA stabilizes this transcript (Casey et al., 1988;Mü llner and Kü hn, 1988;Mü llner et al., 1989;Koeller et al., 1989). The net result of this RNA-protein interaction is thus increased cellular iron uptake and availability. The inverse effect ensues when iron is high, as IRP-1 no longer binds well to the IRE hairpins (reviewed by Kü hn (1994)).A second IRE-binding protein has been characterized in rodents Guo et al., 1994) and in human cells (Samaniego et al., 1994), and is now commonly referred to as IRP-2. IRP-2 is a 105-kDa protein...

Mammalian iron metabolism and its control by iron regulatory proteins

“…Test-tube RNA evolution experiments (SELEX) provided the first indication of the sequence and structure requirements for IRP recognition of IRE elements [125,126,137,138]. In this regard, one of the more intriguing results came from studies where the entire IRE sequence was randomized and IRP1 was used to select high affinity RNA ligands.…”

Iron-responsive element-binding protein 2 plays an essential role in regulating prostate cancer cell growth

“…IRP1 and IRP2 share an overall amino acid identity of approximately 61% [ 5 ] and both have high affinity for IREs. Despite these similarities, IRP1 and IRP2 have been reported to execute different cellular functions through regulation of non-overlapping mRNA targets [ 17 , 21 ]. Our findings support a functional difference between IRP1 and IRP2 by demonstrating that IRP2 is specifically co-opted by prostate cancer cells to alter normal iron homeostasis and retain increased intracellular iron (Figure 2 ).…”

Iron Regulatory Protein 2 Exerts its Oncogenic Activities by Suppressing TAp63 Expression

“…Since p63 3'UTR is responsive to IRP2 (Fig. 3D-E), we searched for a conserved IRE consensus sequence consist of six-membered (CAGUGX) loop that can be recognized by IRP2 (53,54). Interestingly, we located a putative IRE (CAGUGU) at nt 2792 in p63 3'UTR (Fig.…”