Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5-untranslated region (5-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (؉51 to ؉94 from the 5-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (؉101 to ؉146). APP 5-UTR conferred translation was selectively downregulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (⌬4 bases:⌬83AGAG86). Iron regulation of APP mRNA through the APP 5-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce A peptide burden during Alzheimer's disease. The amyloid precursor protein (APP)1 is cleaved into the 40 -42-amino acid A peptides that constitute the main component of the neurotoxic amyloid plaques formed during the progression of Alzheimer's disease (AD) and Down's syndrome (1, 2). In healthy individuals, APP holoprotein is expressed ubiquitously as a protein resembling a type I transmembrane receptor and metal-binding protein (3-6). Secreted APP (APP(s)) is neurotrophic (7).There are now several reports supporting an important role for translational regulatory mechanisms to control APP synthesis and probably A peptide secretion in biologically relevant circumstances (8). First, interleukin-1 (IL-1), the first cytokine released during the acute phase response, significantly increases APP protein synthesis in astrocytes without altering APP mRNA levels (9). IL-1 acts by regulating APP and ferritin genes at the level of message translation (9). Second, reversible ischemic assault significantly increases APP levels without any alteration in the steady-state levels of APP mRNA in rabbit spinal cord neurons (10). Third, APP mRNA 3Ј-UTR sequences located between alternative poly(A) selection sites maintain efficient translation of microinjected APP in Xenopus oocytes and in Chinese hamster ovary transfectants (11).Iron-responsive elements (IREs) are RNA stem loops...
demonstrated recently using immunoelectron microscopy Philadelphia, PA 19104-6148, USA that mRNAs in transit through the nuclear pore complex 1 Corresponding author (NPC) to the cytoplasm are associated directly with shuttling hnRNPs (Visa et al., 1996). Third, an active, Protein import into the nucleus and export from the transferable nuclear export signal (NES) within the nucleus are signal-mediated processes that require shuttling hnRNP A1 protein has been identified (Michael energy. The nuclear transport process about which the et al., 1995a). Fourth, a direct role for the A1 NES in most information is currently available is classical mRNA export has been demonstrated recently. In these nuclear localization signal (NLS)-mediated nuclear experiments, it was shown, using Xenopus oocytes, that import. However, details concerning the signal-mediinjection of saturating amounts of A1 into the nucleus ated export of proteins and RNAs as well as alternative competitively inhibits export of mRNA while a deletion nuclear import pathways are beginning to emerge. An mutant of A1 which lacks a functional NES does not example of this is the heterogeneous nuclear ribo- (Izaurralde et al., 1997), indicating that the A1 NES plays nucleoprotein (hnRNP) A1 protein which, by virtue of a central role in the export of mRNP particles. its M9 domain, is actively exported from the nucleus A1 is the most extensively characterized of the shuttling and imported into the nucleus via a novel pathway hnRNPs. It is composed of two RNP motif RNA-binding mediated by the recently characterized transportin domains (RBDs) as well as a third RNA-binding domain, protein. Here we report that the shuttling hnRNP K the RGG box (Burd and Dreyfus, 1994). A1 typifies a protein contains a novel shuttling domain (termed class of shuttling hnRNPs which require continuous pol KNS) which has many of the characteristics of M9, inII transcription for complete nuclear localization (Piñol-that it confers bi-directional transport across the Roma and Dreyfuss, 1991;Michael et al., 1995b). Transnuclear envelope. KNS-mediated nuclear import is port of A1 is determined by the M9 domain, a 38 amino dependent on RNA polymerase II transcription, and acid sequence located at the carboxy-terminus. M9 was we show that a classical NLS can override this effect.identified initially as the A1 nuclear localization signal Furthermore, KNS accesses a separate import pathway (NLS) as placement of M9 on normally cytoplasmic from either classical NLSs or M9. This demonstrates reporter proteins results in nuclear localization (Siomi and the existence of a third protein import pathway into Dreyfuss, 1995;Weighardt et al., 1995). Interestingly, M9 the nucleus and thereby defines a new type of nuclear also supplies the A1 nuclear export activity. When fused import/export signal.to a protein which is otherwise retained within the nucleus, Keywords: hnRNP K protein/nuclear export/nuclear such as the pentameric core domain of nucleoplasmin, import/shuttling domain/signal M9 can activ...
Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase–M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1–A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.
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