Intragenic regulation of SOCS3 isoforms.

BACKGROUND: Inflammatory reactions are commonly affected by stress responses. Interleukin-6 signalling is part of the inflammatory response and is stringently regulated by the feedback inhibitor SOCS3 expressed in a short and long isoform. Here, we studied the inhibitory potential of the two SOCS3 isoforms. Furthermore, we analysed the regulation of SOCS3 isoform expression and the role of PKR stress kinase signalling in SOCS3 protein expression. METHODS: We performed Western blotting, reporter assays, genetic analyses and manipulations for studying SOCS3 isoform expression and activation of signalling components involved in interleukin-6-induced and PKR-dependent signalling. RESULTS: Interleukin-6-induced endogenous expression of both SOCS3 isoforms was found in distinct cell types. Forced expression of either the long or short SOCS3 isoform demonstrated equal inhibitory activity of each isoform and confirmed longer half-life of the short isoform. Study of intragenic regulation of SOCS3 isoform expression revealed that (i) the 5'-UTR of SOCS3 mRNA restrains specifically expression of the long SOCS3 isoform, (ii) expression of the long isoform restrains expression of the short isoform, and (iii) signalling through the stress kinase PKR does not impact on SOCS3 isoform ratio. CONCLUSIONS: Both SOCS3 isoforms show a similar potential for inhibiting interleukin-6 signalling but differ in their half-lives. Relative expression of the isoforms depends on intragenic elements yet is independent of PKR signalling.

HIV co-opts a cellular antiviral mechanism, activation of stress kinase PKR by its RNA, to enable splicing of rev/tat mRNA.

BACKGROUND: Activation of RNA-dependent stress kinase PKR, especially by viral double-stranded RNA, induces eukaryotic initiation factor 2 α-chain (eIF2α) phosphorylation, attenuating thereby translation. We report that this RNA-mediated negative control mechanism, considered a cornerstone of the cell's antiviral response, positively regulates splicing of a viral mRNA. RESULTS: Excision of the large human immunodeficiency virus (HIV) rev/tat intron depends strictly on activation of PKR by the viral RNA and on eIF2α phosphorylation. Rev/tat mRNA splicing was blocked by viral PKR antagonists Vaccinia E3L and Ebola VP35, as well as by a trans-dominant negative mutant of PKR, yet enhanced by overexpressing PKR. Expression of non-phosphorylatable mutant eIF2αS51A, but not of wild type eIF2α, abrogated efficient splicing of rev/tat mRNA. By contrast, expression of eIF2αS51D, a phosphomimetic mutant of eIF2α, left rev/tat mRNA splicing intact. Unlike eIF2αS51A, eIF2αS51D does not inhibit eIF2α phosphorylation by activated PKR. All HIV mRNA species contain terminal trans-activation response (TAR) stem-loop sequences that potentially could activate PKR, yet even upon TAR deletion, HIV mRNA production remained sensitive to inhibitors of PKR activation. Bioinformatic and mutational analyses revealed a compact RNA pseudoknot upstream of 3'-terminal TAR that promotes splicing by activating PKR. Supporting its essential role in control of splicing, this pseudoknot is conserved among diverse HIV and nonhuman primate SIVcpz isolates. The pseudoknot and 3'-terminal TAR collaborate in mediating PKR-regulated splicing of rev/tat intron, the pseudoknot being dominant. CONCLUSIONS: Our results on HIV provide the first example of a virus co-opting activation of PKR by its RNA, a cellular antiviral mechanism, to promote splicing. They raise the question whether other viruses may use local activation of host kinase PKR through RNA elements within their genome to achieve efficient splicing of their mRNA. Our experiments reveal an indispensable role for eIF2α phosphorylation in HIV rev/tat mRNA splicing that accounts for the need for PKR activation.

Dynamic refolding of IFN-gamma mRNA enables it to function as PKR activator and translation template.

Interferon-gamma mRNA activates the RNA-dependent protein kinase PKR, which in turn strongly attenuates translation of interferon-gamma mRNA. Unlike riboswitches restricted to noncoding regions, the interferon-gamma RNA domain that activates PKR comprises the 5' UTR and 26 translated codons. Extensive interferon-gamma coding sequence is thus dedicated to activating PKR and blocking interferon-gamma synthesis. This implies that the PKR activator is disrupted by ribosomes during translation initiation and must refold promptly to restore PKR activation. The activator structure harbors an essential kink-turn, probably to allow formation of a pseudoknot that is critical for PKR activation. Three indispensable short helices, bordered by orientation-sensitive base pairs, align with the pseudoknot stem, generating RNA helix of sufficient length to activate PKR. Through gain-of-function mutations, we show that the RNA activator can adopt alternative conformations that activate PKR. This flexibility promotes efficient refolding of interferon-gamma mRNA, which is necessary for its dual function as translation template and activator of PKR, and which thus prevents overexpression of this inflammatory cytokine.

Control of mRNA Splicing by Intragenic RNA Activators of Stress Signaling: Potential Implications for Human Disease.

A critical step in the cellular stress response is transient activation of the RNA-dependent protein kinase PKR by double-helical RNA, resulting in down-regulation of protein synthesis through phosphorylation of the α chain of translation initiation factor eIF2, a major PKR substrate. However, intragenic elements of 100-200 nucleotides in length within primary transcripts of cellular genes, exemplified by the tumor necrosis factor (TNF)-α gene and fetal and adult globin genes, are capable of forming RNA structures that potently activate PKR and thereby strongly enhance mRNA splicing efficiency. By inducing nuclear eIF2α phosphorylation, these PKR activator elements enable highly efficient early spliceosome assembly yet do not impair translation of the mature spliced mRNA. The TNF-α RNA activator of PKR folds into a compact pseudoknot that is highly conserved within the phylogeny. Upon excision of ß-globin first intron, the RNA activator of PKR, located in exon 1, is silenced through strand displacement by a short sequence within exon 2, restricting thereby the ability to activate PKR to the splicing process without impeding subsequent synthesis of ß-globin essential for survival. This activator/silencer mechanism likewise controls splicing of α-globin pre-mRNA, but the exonic locations of PKR activator and silencer sequences are reversed, demonstrating evolutionary flexibility. Impaired splicing efficiency may underlie numerous human ß-thalassemia mutations that map to the ß-globin RNA activator of PKR or its silencer. Even where such mutations change the encoded amino acid sequence during subsequent translation, they carry the potential of first impairing PKR-dependent mRNA splicing or shutoff of PKR activation needed for optimal translation.

Control of mRNA splicing by noncoding intragenic RNA elements that evoke a cellular stress response.

Once activated by double-helical RNA, mammalian RNA-dependent stress protein kinase, PKR, phosphorylates its substrate, translation initiation factor eIF2α, to inhibit translation. eIF2α phosphorylation is critical for mounting a cellular stress response. We describe short, 100-200 nucleotide elements within cellular genes that, once transcribed, form RNA structures that potently activate PKR in the vicinity of the RNA and thereby tightly regulate gene expression in cis. Intragenic RNA activators of PKR can (a) attenuate translation of the encoded mRNA by activating PKR and inducing eIF2α phosphorylation, exemplified by the IFN-γ gene, or (b) greatly enhance mRNA splicing efficiency by activating PKR and inducing nuclear eIF2α phosphorylation, thus enabling efficient early spliceosome assembly, exemplified by the adult and fetal globin genes and the TNF-α gene that activates PKR through an RNA pseudoknot conserved from fish to humans. These opposite outcomes considerably extend the potential scope of gene regulation by these novel RNA elements.

An Ancient Pseudoknot in TNF-α Pre-mRNA Activates PKR, Inducing eIF2α Phosphorylation that Potently Enhances Splicing.

Tumor necrosis factor alpha (TNF-α) is expressed promptly during inflammatory responses. Efficient TNF-α mRNA splicing is achieved through a 3' UTR element that activates RNA-dependent eIF2α protein kinase (PKR). The TNF-α RNA activator, we show, folds into a pseudoknot conserved from teleost fish to humans, critical for PKR activation and mRNA splicing. The pseudoknot constrains the RNA into two double-helical stacks having parallel axes, permitting facile PKR dimerization and trans-autophosphorylation needed for kinase activation. Mutations show that the PKR activator potently enhances splicing without inhibiting translation. eIF2α phosphorylation represses translation and is essential for coping with cellular stress, yet PKR-enabled TNF mRNA splicing depends strictly on eIF2α phosphorylation. Indeed, eIF2α phosphorylation at Serine51 is necessary and sufficient to achieve highly efficient splicing, extending its role from negative control of translation to positive control of splicing. This mechanism, operational in human peripheral blood mononuclear cells (PBMCs), links stress signaling to protective immunity through TNF mRNA splicing rendered efficient upon eIF2α phosphorylation.

PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly.

Short elements in mammalian mRNA can control gene expression by activating the RNA-dependent protein kinase PKR that attenuates translation by phosphorylating cytoplasmic eukaryotic initiation factor 2α (eIF2α). We demonstrate a novel, positive role for PKR activation and eIF2α phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35. Splicing and early-stage spliceosome assembly on ß-globin pre-mRNA depend strictly on activation of PKR by a codon-containing RNA fragment within exon 1 and on phosphorylation of nuclear eIF2α on Serine 51. Nonphosphorylatable mutant eIF2αS51A blocked ß-globin mRNA splicing in cells and nuclear extract. Mutations of the ß-globin RNA activator abrogated PKR activation and profoundly affected mRNA splicing efficiency. PKR depletion abrogated splicing and spliceosome assembly; recombinant PKR effectively restored splicing. Excision of the first intron of ß-globin induces strand displacement within the RNA activator of PKR by a sequence from exon 2, a structural rearrangement that silences the ability of spliced ß-globin mRNA to activate PKR. Thus, the ability to activate PKR is transient, serving solely to enable splicing. α-Globin pre-mRNA splicing is controlled likewise but positions of PKR activator and silencer are reversed, demonstrating evolutionary flexibility in how PKR activation regulates globin mRNA splicing through eIF2α phosphorylation.