SIREs: searching for iron-responsive elements

W360–W367 Nucleic Acids Research, 2010, Vol. 38, Web Server issue doi:10.1093/nar/gkq371 Published online 11 May 2010 SIREs: searching for iron-responsive elements Monica Campillos1, Ildefonso Cases2, Matthias W. Hentze1 and Mayka Sanchez1,2,* 1 European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany and 2Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Crta Can Ruti, Camı́ de les Escoles s/n, 08916 Badalona, Barcelona, Spain Received January 23, 2010; Revised April 21, 2010; Accepted April 26, 2010 ABSTRACT INTRODUCTION Post-transcriptional gene regulation including mRNA stability regulation and translational control is an integral part of gene expression and enables more rapid responses and fine-tuning of cell changing conditions (1). The coordinated expression of cellular iron homeostasis by the iron regulatory protein/iron-responsive element regulatory system is among the best characterized posttranscriptional regulatory mechanisms in vertebrates (2). *To whom correspondence should be addressed. Tel: 0034935543077; Fax: 0034934651472; Email: The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The iron regulatory protein/iron-responsive element regulatory system plays a crucial role in the post-transcriptional regulation of gene expression and its disruption results in human disease. IREs are cis-acting regulatory motifs present in mRNAs that encode proteins involved in iron metabolism. They function as binding sites for two related trans-acting factors, namely the IRP-1 and -2. Among cis-acting RNA regulatory elements, the IRE is one of the best characterized. It is defined by a combination of RNA sequence and structure. However, currently available programs to predict IREs do not show a satisfactory level of sensitivity and fail to detect some of the functional IREs. Here, we report an improved software for the prediction of IREs implemented as a user-friendly web server tool. The SIREs web server uses a simple data input interface and provides structure analysis, predicted RNA folds, folding energy data and an overall quality flag based on properties of well characterized IREs. Results are reported in a tabular format and as a schematic visual representation that highlights important features of the IRE. The SIREs (Search for iron-responsive elements) web server is freely available on the web at http://ccbg.imppc.org/sires/index.html This system involves two cytoplasmic iron regulatory proteins, IRP1 and IRP2, and RNA stem–loop, known as IREs, within transcripts encoding iron metabolism proteins. Under conditions of iron starvation, IRPs bind to the IREs and control the expression of target mRNAs by two different mechanisms. Either of the IRPs induces translational repression when bound to an IRE located at the 50 UTR, whereas their association with IREs in the 30 UTR mediates mRNA stabilization (3,4). The central role of the IRPs in iron homeostasis is highlighted by the observation that total and constitutive genetic ablation of both IRP1 and IRP2 causes embryonic lethality in mice (5). Furthermore, tissue-specific disruption of both IRPs in duodenal enterocytes revealed that these proteins are essential for intestinal function (6). IREs have been reported in a total of 12 mRNAs, 7 containing an IRE in their 50 UTRs and 5 in their 30 UTRs. 50 UTR IREs include those present in the mRNAs coding for the iron storage proteins ferritin L (FTL) and ferritin H (FTH1; 7), the heme biosynthesis enzyme ALAS2 (8), the iron exporter ferroportin (SLC40A1; 9), two enzymes of the citric acid cycle (mitochondrial aconitase ACO2 and Drosophila melanogaster succinate dehydrogenase dSDH; 10) and the transcription factor and oxygen sensor EPAS1 (also known as HIF2alpha; 11). 30 UTR IREs have been identified in mRNAs for iron acquisition molecules (TFR1 and SLC11A2; 12,13), the human cell-cycle phosphatase CDC14A (14), the human myotonic dystrophy kinaserelated Cdc42-binding kinase alpha (CDC42BPA; 15) and the mouse glycolate oxidase (Hao1; 16). In humans, the failure to coordinate the expression of IRE-containing genes is associated with pathologic conditions, as illustrated by the autosomal dominant hyperferritinemiacataract syndrome observed in patients carrying mutations in the FTL IRE (17; HHCS, OMIM 600886), or by an autosomal dominant iron overload syndrome associated with a mutation in the FTH1 IRE (18; OMIM 134770). A canonical IRE structure is composed of a 6-nt apical loop (50 -CAGWGH-30 ; whereby W stands for A or U and Nucleic Acids Research, 2010, Vol. 38, Web Server issue W361 MATERIALS AND METHODS IRE prediction algorithm The SIREs algorithm is implemented on a Perl script that screens for a 19 or 20 nucleotide sequence motif corresponding to the core sequence of an IRE (positions n07–n25) that includes the hexa-apical hairpin loop (n14–n19), the upper stem, the cytosine bulge (C8) and the lower base pair (n07–n25) (Figure 1A). This core IRE region is sufficient to establish the recently reported RNA binding hierarchy between IRP1 and 50 IREs (27). We used a highly specific rule-based decision tree shown in Figure 1B to screen for IRE motifs in nucleotide sequences. First, the sequences are screened to find one of the motifs described in Figure 1 (motif 1–18). These 18 motifs are based on two canonical IRE motifs 50 CNNNNNCAGUGN-30 (motif 1) and 50 -CNNNNNCA GAGN-30 (motif 2) and 16 SELEX (systematic evolution of ligands by exponential enrichment) motifs proven to bind IRP1 and/or IRP2 in vitro with a relative binding efficiency >20% (21–23). All motifs but motif 18 contain a cytosine at position n8; motif 18 has a guanine. Next, pairing of the upper stem nucleotides is tested allowing six pairing combinations (four Watson–Crick base pairs: A–U, U–A, C–G, G–C, and two wobble base pairs: U.G and G.U). The number of G.U or U.G wobble base pairs in the upper stem and at position n07–n25 is limited to a maximum of two, since the presence of three or more wobble base pairs impairs the formation of a proper IRE (data not shown). The SIREs program allows the detection of IRE-like motifs with one mismatch in the upper stem (positions n13–n20, n12– n21, n11–n22, n10–n23, n09–n24) or at position n07– n25, in order to detect IREs like the one present in the Hao1 mRNA, which contains an A:A mismatch at position n11–n22. Similarly, a single bulge in the 30 half of the upper stem (30 bulge) is allowed at positions n20b, n21b, n22b or n23b to detect IREs such as the ones present in the mRNA of (...truncated)