STAU1 binding 3′ UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown

Department of Biochemistry and Biophysics, School of Medicine and Dentistry.
Genes & development (Impact Factor: 10.8). 07/2013; 27(13):1495-510. DOI: 10.1101/gad.220962.113
Source: PubMed


For a number of human genes that encode transcripts containing inverted repeat Alu elements (IRAlus) within their 3' untranslated region (UTR), product mRNA is efficiently exported to the cytoplasm when the IRAlus, which mediate nuclear retention, are removed by alternative polyadenylation. Here we report a new mechanism that promotes gene expression by targeting mRNAs that maintain their 3' UTR IRAlus: Binding of the dsRNA-binding protein Staufen1 (STAU1) to 3' UTR IRAlus inhibits nuclear retention so as to augment the nuclear export of 3' UTR IRAlus-containing mRNAs (IRAlus mRNAs). Moreover, we found that 3' UTR IRAlus-bound STAU1 enhances 3' UTR IRAlus mRNA translation by precluding protein kinase R (PKR) binding, which obviates PKR activation, eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, and repression of global cell translation. Thus, STAU1 binding to 3' UTR IRAlus functions along with 3' UTR IRAlus-mediated nuclear retention to suppress the shutdown of cellular translation triggered by PKR binding to endogenous cytoplasmic dsRNAs. We also show that a changing STAU1/PKR ratio contributes to myogenesis via effects on the 3' UTR IRAlus of mRNA encoding the microRNA-binding protein LIN28.

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    • "Furthermore, although our data support the view that the reduced binding capability of methylated p54 nrb to dsRNAs could result from a direct conformational change of p54 nrb methylation at the coiled-coil domain, we cannot exclude the possibility that these methyl sites may recruit other effector proteins to facilitate the release of mRNA-IRAlus from methylated p54 nrb (Yang et al. 2014). Finally, the dsRNA-binding protein STAU1 (Wickham et al. 1999) was recently shown to compete with p54 nrb for the binding of 3 ′ UTR IRAlus, independent of editing (Elbarbary et al. 2013). It will be of interest to examine whether the binding of 3 ′ UTR IRAlus with STAU1 occurs after the release of mRNAs containing IRAlus from methylated p54 nrb . "
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    ABSTRACT: In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1. © 2015 Hu et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 03/2015; 29(6):630-45. DOI:10.1101/gad.257048.114 · 10.80 Impact Factor
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    • "Changes in the miRNA profile induced by WNV were rather consistent between cell types and the WNV miRNA signature was not significantly affected by the presence or absence of TLR3. These data further suggest that the majority of WNV-regulated miRNAs at early times post-infection (8 hrs) are not TLR3-dependent, but may depend on signaling through other PRRs such as RIG-I and MDA5 and/or other sensors like PKR (RIG-I, MDA5 and PKR are fully functional in HEK293 cells) [71], [72], [73], [74], [75], [76], [77], [78], [79]. "
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    ABSTRACT: The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.
    PLoS ONE 08/2014; 9(8):e104770. DOI:10.1371/journal.pone.0104770 · 3.23 Impact Factor
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    • "This competition between STAU and PKR might account for the lack of pPKR in the nucleus of interphase cells despite the presence of unphosphorylated PKR, as shown in Figure 1E. Consequently, STAU knockdown resulted in increased phosphorylation of PKR and global suppression of translation (Elbarbary et al. 2013). Hence, a low level of pPKR might be present in the cytosol of interphase cells, which can account for the small increase in general translation when PKR is knocked down in interphase (Fig. 5F). "
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    ABSTRACT: dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as CYCLINS A and B and POLO-LIKE KINASE 1 and phosphorylation of HISTONE H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells.
    Genes & Development 06/2014; 28(12):1310-22. DOI:10.1101/gad.242644.114 · 10.80 Impact Factor
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