Identification of Binding Sites for Both dsRBMs of PKR on Kinase-Activating and Kinase-Inhibiting RNA Ligands

Spanggord, Richard J.; Vuyisich, Momchilo; Beal, Peter A.

doi:10.1021/bi0120594

Cited by 46 publications

(53 citation statements)

References 40 publications

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“…RNA molecules that bind to the dsRNA-binding motifs (dsRBMs) that constitute the dsRBD of PKR can either function as activators or inhibitors of the protein kinase, depending on the RNA concentration and whether they interact with one or both motifs (Spanggord et al 2002). We therefore investigated whether the HCV IRES possesses either of these activities.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Vyas

Elia²,

Clemens³

2003

RNA

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Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5 untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA f -binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

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Section: Resultsmentioning

confidence: 99%

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Vyas

Elia²,

Clemens³

2003

RNA

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“…In particular, cleavage affinity assays show that binding of PKR to dsRNAs is mediated by either one or both dsRBDs depending on the ligand, and each dsRDB can recognize different sites on the same RNA 5 (a, d, and g) and an anti-ILF3 antibody (b, e, and h). The localization of the nucleus was drawn in panels c, f, and i. molecule (34). In vivo, ADAR1 and ADAR2 mediate site-specific editing of few cellular RNAs, and both enzymes modify the glutamate receptor B subunit transcript but on distinct adenosines (33,35,36).…”

Section: Discussionmentioning

confidence: 99%

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Gwizdek

Ossareh-Nazari

Brownawell

et al. 2004

Journal of Biological Chemistry

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The karyopherin-related nuclear transport factor exportin-5 preferentially recognizes and transports RNAs containing minihelix motif, a structural cis-acting export element that comprises a double-stranded stem (>14 nucleotides) with a base-paired 5 end and a 3-8-nucleotide protruding 3 end. This structural motif is present in various small cellular and viral polymerase III transcripts such as the adenovirus VA1 RNA (VA1). Here we show that the double-stranded RNA-binding protein, ILF3 (interleukin enhancer binding factor 3) preferentially binds minihelix motif. Gel retardation assays and glutathione S-transferase pull-down experiments revealed that ILF3, exportin-5, RanGTP, and VA1 RNA assembled in a quaternary complex in which the RNA moiety bridges the interaction between ILF3 and exportin-5. Formation of this complex is facilitated by the ability of both exportin-5 and ILF3 to mutually increase their apparent affinity for VA1 RNA. Using microinjection in the nucleus of HeLa cells and transfection experiments, we show here that formation of the cooperative RanGTP-dependent RNA/ILF3/exportin-5 complex promotes the co-transport of VA1 and ILF3 from the nucleus to the cytoplasm. Exportin-5 thus appears as the first example of a nuclear export receptor that mediates RNA export but also promotes transport of proteinaceous cargo through appropriate and specific RNA adaptors.Transfer of macromolecules through nuclear pore complexes depends on both structural components of nuclear pore complexes and soluble factors that ensure nuclear transport of a wide diversity of cellular proteins and RNAs. Selectivity of transport is mediated by the recognition of specific signals within the cargoes by specific transport receptors through a direct or adaptor-mediated interaction. Transport receptors interact with nucleoporins thereby mediating docking to and translocation through the nuclear pore complexes. Most of the transport receptors belong to the karyopherin ␤ family (also known as importin ␤ family) and use the GTPase Ran to control cargo association. The asymmetric distribution of the Ran regulatory proteins provides a steep gradient of RanGDP (cytoplasmic)/RanGTP (nuclear) across the nuclear envelope that ensures the directionality of nuclear transport (1, 2). Indeed, nuclear import receptors (importins) bind their cargoes in the cytoplasm in the absence of RanGTP and unload them upon binding to RanGTP in the nucleus. In contrast, export receptors (exportins) require RanGTP to interact with their cargoes in the nucleus, and those export complexes are destabilized by dissociation of RanGTP and GTP hydrolysis in the cytoplasm (3, 4).Study of nuclear export pathways of diverse viral RNAs has greatly contributed to the identification of cellular factors implicated in nuclear transport processes. In particular, we recently reported nuclear export mechanisms of the adenovirus VA1 RNA (5, 6). This RNA is abundantly transcribed by RNA polymerase III and efficiently exported to the cytoplasm in the late phase of the viral infect...

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“…Manche et al (228) showed that while a duplex sequence of 11 bp could bind to PKR, 33 bp was the minimum length for activation, and maximal activation was achieved with 80 bp. Furthermore, simultaneous binding of PKR to both dsRNA-binding domains is required for its activation (356). This conclusion, however, needs to be tempered by the observation that at least some short dsRNAs can also induce the PKR pathway in a concentration-dependent manner (see below, in the RNAi section).…”

Section: Long Cytoplasmic Dsrnas Trigger Nonspecific and Global Effectsmentioning

confidence: 99%

Effects of Length and Location on the Cellular Response to Double-Stranded RNA

Wang

Carmichael

2004

Microbiol Mol Biol Rev

106

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Since double-stranded RNA (dsRNA) has not until recently generally been thought to be deliberately expressed in cells, it has commonly been assumed that the major source of cellular dsRNA is viral infections. In this view, the cellular responses to dsRNA would be natural and perhaps ancient antiviral responses. While the cell may certainly react to some dsRNAs as an antiviral response, this does not represent the only response or even, perhaps, the major one. A number of recent observations have pointed to the possibility that dsRNA molecules are not seen only as evidence of viral infection or recognized for degradation because they cannot be translated. In some instances they may also play important roles in normal cell growth and function. The purpose of this review is to outline our current understanding of the fate of dsRNA in cells, with a focus on the apparent fact that their fates and functions appear to depend critically not only on where in the cell dsRNA molecules are found, but also on how long they are and perhaps on how abundant they are

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Identification of Binding Sites for Both dsRBMs of PKR on Kinase-Activating and Kinase-Inhibiting RNA Ligands

Cited by 46 publications

References 40 publications

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Effects of Length and Location on the Cellular Response to Double-Stranded RNA

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