Nuclear-cytoplasmic shuttling of APC regulates beta-catenin subcellular localization and turnover.

Mutational inactivation of the APC gene is a key early event in the development of familial adenomatous polyposis and colon cancer. APC suppresses tumour progression by promoting degradation of the oncogenic transcriptional activator beta-catenin. APC gene mutations can lead to abnormally high levels of beta-catenin in the nucleus, and the consequent activation of transforming genes. Here, we show that APC is a nuclear-cytoplasmic shuttling protein, and that it can function as a beta-catenin chaperone. APC contains two active nuclear export sequences (NES) at the amino terminus, and mutagenesis of these conserved motifs blocks nuclear export dependent on the CRM1 export receptor. Treatment of cells with the CRM1-specific export inhibitor leptomycin B shifts APC from cytoplasm to nucleus. beta-catenin localization is also regulated by CRM1, but in an APC-dependent manner. Transient expression of wild-type APC in SW480 (APCmut/mut) colon cancer cells enhances nuclear export and degradation of beta-catenin, and these effects can be blocked by mutagenesis of the APC NES. These findings suggest that wild-type APC controls the nuclear accumulation of beta-catenin by a combination of nuclear export and cytoplasmic degradation.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants.

BACKGROUND: The vast majority of BRCA1 missense sequence variants remain uncharacterized for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. OBJECTIVE: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. METHODS AND RESULTS: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. CONCLUSIONS: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Transcriptional and posttranscriptional activation of urokinase plasminogen activator gene expression in metastatic tumor cells.

Urokinase plasminogen activator (uPA) is a serine protease which has frequently been implicated in the process of tumor cell invasion and metastasis. The degree of expression and mode(s) of regulation of the uPA gene in metastatic compared with nonmetastatic tumor cells have not yet been addressed. We have cloned and sequenced a full-length rat uPA complementary DNA and utilized Northern blot analysis to report that the uPA gene is expressed at levels 3.5- to 70-fold higher in metastatic cell lines than in nonmetastatic cell lines derived from two independent rat mammary adenocarcinomas. Nuclear run-on assays and RNA half-life estimations indicated that metastatic MAT 13762 rat mammary adenocarcinoma cells expressed 3.5-fold higher levels of uPA RNA than a nonmetastatic derivative (J-clone), due to a combined increase in uPA gene transcription and cytoplasmic RNA stability. By contrast, uPA RNA (and enzyme) levels were elevated by up to 70-fold in metastatic clones of dimethylbenz(a)anthracene-induced rat mammary adenocarcinoma (DMBA-8) due to predominantly posttranscriptional mechanisms. Moreover, treatment of nonmetastatic DMBA-8 cell lines with protein synthesis inhibitors led to an increase in nuclear and cytoplasmic uPA RNA levels, without altering the rate of uPA gene transcription. These results suggest that in addition to gene transcription, posttranscriptional events localized in the nucleus and cytoplasm are key determinants of uPA gene activation in rat mammary adenocarcinomas.

Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta.

The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can "shuttle" between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-beta. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-beta did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-alpha/beta heterodimer, the binding of Rev to importin-beta was actually blocked by importin-alpha receptor. Our findings suggest that Rev and importin-alpha bind (via an arginine-rich sequence) to a similar region on importin-beta. In addition, we show that the complex between Rev and importin-beta can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.

Inhibition of ANKRD1 sensitizes human ovarian cancer cells to endoplasmic reticulum stress-induced apoptosis.

High expression of Ankyrin Repeat Domain 1 (ANKRD1) in ovarian carcinoma is associated with poor survival, and in ovarian cancer cell lines is associated with platinum resistance. Importantly, decreasing ANKRD1 expression using siRNA increases cisplatin sensitivity. In this study, we investigated possible mechanisms underlying the association of ANKRD1 with cisplatin response. We first demonstrated that cisplatin-induced apoptosis in ovarian cancer cell lines was associated with endoplasmic reticulum (ER) stress, evidenced by induction of Glucose-Regulated Protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and increased intracellular Ca(2+) release. The level of sensitivity to cisplatin-induced apoptosis was associated with ANKRD1 protein levels and poly (ADP-ribose) polymerase (PARP) cleavage. COLO 316 ovarian cancer cells, which express high ANKRD1 levels, were relatively resistant to cisplatin, and ER stress-induced apoptosis, whereas OAW42 and PEO14 cells, which express lower ANKRD1 levels, are more sensitive to ER stress-induced apoptosis. Furthermore, we show that overexpression of ANKRD1 attenuated cisplatin-induced cytotoxicity, and conversely siRNA knockdown of ANKRD1 sensitized ovarian cancer cells to cisplatin and ER stress-induced apoptosis associated with induction of GADD153, and downregulation of BCL2 and BCL-XL. Taken together, these results suggest that ANKRD1 has a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy.

Interaction between iron-regulatory proteins and their RNA target sequences, iron-responsive elements.

In this chapter, we have focused on the biochemistry of IRP-1 and the features which distinguish it from the related RNA-binding protein, IRP-2. IRP-1 is the cytoplasmic isoform of the enzyme aconitase, and, depending on iron status, may switch between enzymatic and RNA-binding activities. IRP-1 and IRP-2 are trans-acting regulators of mRNAs involved in iron uptake, storage and utilisation. The finding of an IRE in the citric acid cycle enzymes, mitochondrial aconitase and succinate dehydrogenase, suggests that the IRPs may also influence cellular energy production. These two proteins appear to bind RNAs with different but overlapping specificity, suggesting that they may regulate the stability or translation of as yet undefined mRNA targets, possibly extending their regulatory function beyond that of iron homeostasis. The interaction between the IRPs and the IRE represents one of the best characterised model systems for posttranscriptional gene control, and given that each IRP can also recognise its own unique set of RNAs, the search for new in vivo mRNA targets is expected to provide yet more surprises and insights into the fate of cytoplasmic mRNAs.

TATA box-independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes.

Transcription of the human tissue-type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif [1985, J. Biol. Chem. 260, 11223-11230]. In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol-12-myristate 13-acetate (PMA) induced WI-38 human lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA-proximal location (+1), a 10-fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA-independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5'-CAGAGCTG-3') was identified which is common to the TATA-independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA-independent genes.

Expression levels of heat shock protein 60 in human endothelial cells in vitro are unaffected by exposure to 50 Hz magnetic fields.

Magnetic fields (MFs) from domestic power sources have been implicated as being a potential risk to human health. A number of epidemiological studies have found a significant link between exposure to MFs and increased rates of cancers. There have also been a number of in vivo and in vitro studies reporting effects of MFs in animal disease models and on the expression or activity of a range of proteins. In the past decade, our group proposed that atherosclerosis may have an autoimmune component, with heat shock protein 60 (Hsp60) expressed in endothelial cells as the dominant autoantigen. A number of stressors have been shown to induce the expression of Hsp60, including the classical risk factors for atherosclerosis. We were interested to see if the exposure of endothelial cells to an MF elicited increased expression of Hsp60, as has been reported previously for Hsp70. The present work describes the exposure of endothelial cells to domestic power supply (50 Hz) MFs at an intensity of 700 microT. The results from our system indicate that cultured endothelial cells exposed to a high intensity of MF either alone or in combination with classical heat stress show no effects on the expression of Hsp60 at either the messenger ribonucleic acid or the protein level. As such, there is no evidence that exposure to extremely low-frequency MF would be expected to increase the expression of Hsp60 and therefore the initiation or progression of atherosclerosis.

The management of unilateral lateral mass/facet fractures of the subaxial cervical spine: the use of magnetic resonance imaging to predict instability.

STUDY DESIGN: Retrospective review of the clinical course and cervical spine plain radiographs, computed tomography, and magnetic resonance imaging of 24 consecutive patients for a 2-year period with a unilateral lateral mass/facet fracture. OBJECTIVE: To propose a treatment algorithm for the management of unilateral lateral mass/facet fractures of the subaxial cervical spine based on ligamentous injury detected by magnetic resonance imaging. SUMMARY OF BACKGROUND DATA: There have been no previous reports of the use of magnetic resonance imaging to predict clinical instability. METHODS: A retrospective review of the clinical course of all unilateral mass/facet fractures identified over a 2-year period was conducted. All cervical spine plain radiographs, computed tomography scans, and magnetic resonance images were reviewed by a neuroradiologist blinded to the clinical course of the patient. Magnetic resonance T1-weighted and inversion recovery images were used to evaluate the integrity of the facet region, interspinous ligament, anterior longitudinal ligament, and posterior longitudinal ligament. RESULTS: Twenty-four unilateral lateral mass/facet fractures were identified. Only six initial cervical spine series demonstrated a bony abnormality at the level of the fracture. The fractures were identified by computed tomography and were almost all nondisplaced or minimally displaced. Less than half of the fractures extended ventrally to involve the transverse process or foramen transversarium or dorsally to involve the lamina. Twelve fractures were nonoperatively treated and 12 were treated surgically for stabilization. Ten patients in the operative group presented with or developed a subluxation. Nine of these patients had injury to at least three of the four ligaments evaluated by magnetic resonance imaging. In the nonoperative group, only three patients had extensive ligamentous injury at the level of the fracture. All three of these patients were lost to follow-up. CONCLUSIONS: Plain radiographs of the cervical spine lack sensitivity to detect the presence of lateral mass/ facet fractures. The appearance of the fracture on computed tomography does not indicate instability. The degree of ligamentous injury at the level of the fracture demonstrated on magnetic resonance imaging correlates with instability in this series. Operative stabilization may be indicated for unilateral lateral mass fractures that present with a subluxation or that have injury to at least three of the following ligaments: the facet region, the interspinous ligament, the anterior longitudinal ligament, and the posterior longitudinal ligament. However, before a definitive management plan can be formulated, results from this small series require further validation.

Expression of the nm23-2/NDP kinase alpha gene in rat mammary and oral carcinoma cells of varying metastatic potential.

Reduced expression of the putative metastasis-suppressor gene, nm23-1, has previously been correlated with high tumour metastatic potential. The involvement of the related and proximally-located nm23-2 gene in the suppression of tumour metastasis, however, has not yet been tested. In this study, we compared nm23-2 RNA levels in cell lines derived from three independent rat mammary carcinomas. Northern blot analysis revealed no correlation between nm23-2 RNA levels and metastatic potential in parent or clonal cell lines derived from chemically-induced (MAT 13762, DMBA-8) or spontaneous (BC1) rat mammary carcinomas. Cloning and sequencing of an nm23-2 cDNA from metastatic BC1 cells demonstrated that the predicted coding sequence of nm23-2 RNA in these cells was not inactivated by mutation. Further analysis showed that the nm23-2 gene was not down-regulated in H-ras-transfected metastatic clones or other metastatic cell lines derived from a spontaneous rat paraoral squamous cell carcinoma, B10. The data do not suggest a correlation between nm23-2 gene expression and metastasis-suppression in these tumours.

Iron regulatory proteins 1 and 2.

Iron uptake and storage in mammalian cells is at least partly regulated at a post-transcriptional level by the iron regulatory proteins (IRP-1 and IRP-2). These cytoplasmic regulators share 79% similarity in protein sequence and bind tightly to conserved mRNA stem-loops, named iron-responsive elements (IREs). The IRP:IRE interaction underlies the regulation of translation and stability of several mRNAs central to iron metabolism. The question of why the cell requires two such closely related regulatory proteins may be resolved as we learn more about the expression and regulation of these proteins. It is evident so far that, despite similarities, the IRPs differ in several important respects. They are coordinately regulated by cellular iron, but whereas IRP-1 is inactivated by high iron levels, IRP-2 is rapidly degraded. Further differences arise in their expression and RNA-binding specificity. The two proteins each recognise a large repertoire of IRE-like sequences, including a small group of exclusive RNA targets. These findings hint that IRP-1 and IRP-2 may bind preferentially to certain mRNAs in vivo, possibly extending their known functions beyond the regulation of intracellular iron homeostasis.

A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals.

Nuclear export sequences (NESs) have been identified in many cellular proteins, but it remains unclear how different NESs compare in activity. We describe a sensitive new in vivo export assay which we have used to assess the relative export activity of different types of NES. The most common type of export sequence resembles that first identified in the HIV-1 Rev protein and typically comprises a core of large hydrophobic amino acids that specify recognition by the CRM1 export receptor. We compared 10 previously identified Rev-type NESs in our assay, and whereas all were functional, the relative export activities of these signals varied considerably. We further identified 3 new Rev-type NESs from a computer database search, and each export signal was assigned a score of 1 to 9 and ranked in order of activity (e.g., PKI > c-ABL > Ran-BP1 > FMRP > PML > IkappaB-alpha > hdm2). The weakest NESs were found in the p53 tumor suppressor and the p53-regulated proteins p21 and hdm2, which are all normally localized to the nucleus. All of the Rev-type NESs were inactivated by mutation of key hydrophobic residues and by treatment with the CRM1-specific export inhibitor, leptomycin B. In contrast, a different type of export signal, the KNS shuttling element derived from hnRNP K, exhibited a modest export activity that was insensitive to leptomycin B treatment. KNS thus appears to mediate export via a CRM1-independent pathway. Mutagenesis of the KNS sequence identified, for the first time, specific serines and acidic residues necessary for its export activity, thereby distinguishing KNS from other types of nuclear transport signal. We have shown that different nuclear export signals can vary profoundly in activity and therefore conclude that the nuclear export rate of a specific shuttling protein largely depends on both the strength and the accessibility of its NES.

Differential modulation of the RNA-binding proteins IRP-1 and IRP-2 in response to iron. IRP-2 inactivation requires translation of another protein.

Iron regulatory proteins (IRPs)-1 and -2 bind specific mRNA hairpin structures known as iron-responsive elements and thereby post-transcriptionally regulate proteins involved in iron uptake, storage, and utilization. In this study, we compared modulation of the RNA-binding activities of IRP-1 and IRP-2. We show that in vitro RNA-binding can be inhibited for each IRP by the alkylation of free sulfhydryl groups with N-ethylmaleimide, or by oxidation with diamide. The in vivo iron regulation of IRP-1 and IRP-2 appeared to involve different pathways. Both proteins are activated in Ltk- cells following iron chelation. This induction, however, was distinguishable by the addition of translation inhibitors, which temporarily delayed activation of IRP-1 by up to 8 h, but fully blocked IRP-2 induction for up to 20 h. The activation of IRP-2 was also prevented by transcription inhibition with actinomycin D. Further analysis revealed that, while both IRPs are rapidly inactivated following iron treatment of iron-depleted cells, the repression of IRP-2 was again completely translation dependent. Immunoblot analysis suggests that iron modulation of IRP-1 activity is predominantly a posttranslational process. This contrasts with IRP-2, whose activation reflected the accumulation of stable IRP-2 protein by de novo synthesis. IRP-2 inactivation/degradation occurred upon readdition of iron, but it required translation of another protein. The existence of an independent regulator of IRP-2 may help explain the differential regulation and expression of the two IRP proteins in different tissues and cell lines.

Identification of a functional nuclear export sequence in BRCA1.

Germ-line mutations in the tumor suppressor gene Brca1 confer increased susceptibility to breast and ovarian cancers. BRCA1 is a 1863-amino acid protein with roles in transcriptional regulation and the cellular responses to DNA damage. Given its function in these nuclear processes, the subcellular localization of BRCA1 is an important issue and has been the object of recent controversy. BRCA1 contains two nuclear localization signals and is most frequently detected in the cell nucleus by immunofluorescence microscopy. In this study, we show that BRCA1 is a nuclear-cytoplasmic shuttling protein, capable of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export sequence ((81)QLVEELLKIICAFQLDTGL) near the amino terminus of BRCA1 that facilitates export via the CRM1/exportin pathway. Mutational inactivation of this nuclear export sequence, or treatment of cells with the CRM1-specific export inhibitor leptomycin B, induced nuclear accumulation of ectopic full-length BRCA1. Moreover, overexpression of the CRM1 export receptor resulted in decreased nuclear localization of endogenous BRCA1. The unexpected ability of BRCA1 to shuttle between nucleus and cytoplasm may have implications for the regulation and function of this tumor suppressor.

Dexamethasone decreases urokinase plasminogen activator mRNA stability in MAT 13762 rat mammary carcinoma cells.

The glucocorticoid dexamethasone was observed to decrease urokinase plasminogen activator (uPA) RNA levels from within 1 h of treatment of MAT 13762 mammary adenocarcinoma cells. The drug did not alter the rate of uPA gene transcription in these cells, but decreased the stability of cytoplasmic uPA mRNA transcripts. Results from cycloheximide and actinomycin D experiments indicated that the dexamethasone-mediated reduction in uPA RNA required both new protein and RNA synthesis. Based on these results, we propose that dexamethasone induces a short-lived protein(s) which down-regulates uPA RNA levels post-transcriptionally in these metastatic tumour cells.

Post-transcriptional regulation of urokinase plasminogen activator gene expression occurs in the nucleus of BC1 rat mammary tumor cells.

The regulation of urokinase plasminogen activator (uPA) expression was investigated in 2 highly metastatic rat mammary adenocarcinoma cell lines, BC1 and MAT 13762. BC1 cells were observed to synthesize, on average, 10 times less uPA enzyme and mRNA than MAT 13762 cells; however this difference was not accounted for by differences in uPA gene copy number/structure or in the rate of uPA gene transcription in the cell lines studied. Moreover, Northern blot analysis of invasive sub-populations derived in vitro from the BC1 cell line revealed levels of uPA expression similar to those of the parent, but a 3-fold elevation in expression of the metalloprotease gene, transin. Further investigation showed that treatment of BC1 cells with either of the protein synthesis inhibitors, cycloheximide or anisomycin, increased the level of both nuclear and cytoplasmic uPA RNA 6- to 18-fold in 4 hr, whilst inducing a maximum 2.6-fold increase in the rate of uPA gene transcription. This increase in uPA gene expression may therefore reflect, in part, an increase in the stability and/or processing of nuclear uPA transcripts. These results suggest that the degree of uPA gene expression does not correlate directly with BC1 tumor-cell invasion in vitro, and that the uPA gene is down-regulated, at least in part, post-transcriptionally in the nucleus of BC1 mammary tumor cells.

Characterization of a second RNA-binding protein in rodents with specificity for iron-responsive elements.

Iron regulatory factor (IRF) is a cytoplasmic RNA-binding protein involved in regulating iron homeostasis. IRF controls expression of ferritin and transferrin receptor post-transcriptionally via specific binding to stem-loop iron-responsive elements (IREs) located in the untranslated regions of the respective mRNAs. We have confirmed by RNA band-shift analysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulated by intracellular iron levels. This faster migrating complex appears to represent a specific interaction between the ferritin IRE and an iron-regulated protein that is distinct from IRF, as concluded from the following lines of evidence. First, UV cross-linking and partial digestion with different proteases revealed different peptide patterns for the two IRE-protein complexes. Second, antiserum raised against IRF peptides immunoprecipitated only authentic IRF and not the protein of the faster migrating complex, as determined by band-shift analysis. Following separation of the two IRE-binding proteins by ion-exchange chromatography, only the IRF-containing fraction reacted with the antibodies on Western blots. The second protein binds IREs with an affinity similar to that of IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. UV cross-linking experiments indicate that this second protein, tentatively named IRFB, has a molecular mass of approximately 105 kDa. Analysis of mouse tissues revealed differences in the distribution of IRF and IRFB. Whereas IRF protein and IRE binding activity were predominant in liver, intestine, and kidney, the IRFB protein(s) revealed highest binding activity in intestine and brain. Our data support the existence of two distinct iron-regulated IRE-binding proteins in rodents.

Iron regulatory proteins 1 and 2 bind distinct sets of RNA target sequences.

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.

Succinate dehydrogenase b mRNA of Drosophila melanogaster has a functional iron-responsive element in its 5'-untranslated region.

Iron-responsive elements (IREs) are cis-acting mRNA stem-loop structures that specifically bind cytoplasmic iron regulatory proteins (IRPs). IRP-IRE interactions mediate the coordinate post-transcriptional regulation of key proteins in iron metabolism, such as ferritin, transferrin receptor, and erythroid 5-aminolevulinic acid synthase. Depending on whether the IRE is located in the 5'- or 3'-untranslated region (UTR), binding of IRP will inhibit mRNA translation or degradation, respectively. Here we describe a new IRE in the 5'-UTR of succinate dehydrogenase subunit b (SDHb) mRNA of Drosophila melanogaster. The SDHb IRE binds in vitro to vertebrate and insect IRPs with a high affinity equal to that of human ferritin H chain IRE. Under conditions of iron deprivation, SDHb mRNA of Drosophila SL-2 cells shifts to a non-polysome-bound pool. Moreover, translation of a human growth hormone mRNA with the SDHb IRE in its 5'-UTR is iron-dependent in stably transfected L cells. We conclude that the SDHb IRE mediates translational inhibition both in insect and vertebrate cells. This constitutes the first identification of a functional IRE in insects. Furthermore, Drosophila SDHb represents the second example, after porcine mitochondrial aconitase, of an enzyme of the citric acid cycle whose mRNA possesses all necessary features for translational regulation by cellular iron levels.