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.

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.

Early changes in metabolism of leukemic cell lines upon induction of apoptosis by cytotoxic drugs.

We evaluated real-time changes in extracellular acidification rates of human U937 and K562 leukemic cells treated with camptothecin or taxol. U937 cells treated with camptothecin or taxol for 30-60 min showed a continuous, irreversible decrease in extracellular acidification rate that was sensitive to amiloride. In contrast, U937 cells exposed to sodium azide showed an immediate, steep decrease in extracellular acidification rate that was reversible upon azide withdrawal. K562 cells required a >20-fold higher dose of camptothecin to promote similar changes in the extracellular acidification rate, with a corresponding resistance in their susceptibility to camptothecin- or taxol-induced apoptosis. The data show that irreversible commitment to apoptosis is associated with rapid metabolic changes that are reflected by decreased extracellular acidification rate and regulated by the Na(+)/H(+) antiporter. Moreover, detection of extracellular acidification rate changes was not restricted to a particular cell type or apoptosis pathway, making this a potentially useful tool to screen compounds for pro-apoptotic activity.

Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR.

PKR, an interferon (IFN)-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2alpha chain. We show that human IFN-gamma mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-gamma mRNA activates PKR through a pseudoknot in its 5' untranslated region. Mutations that impair pseudoknot stability reduce the ability to activate PKR and strongly increase the translation efficiency of IFN-gamma mRNA. Nonphosphorylatable mutant eIF2alpha, knockout of PKR and PKR inhibitors 2-aminopurine, transdominant-negative PKR, or vaccinia E3L correspondingly enhances translation of IFN-gamma mRNA. The potential to form the pseudoknot is phylogenetically conserved. We propose that the RNA pseudoknot acts to adjust translation of IFN-gamma mRNA to the PKR level expressed in the cell.