Activation of the double‐stranded RNA‐dependent eIF‐2α kinase by cellular RNA from 3T3‐F442A cells

Abstract

The interferon induced double-stranded-RNA-dependent eIF-2a kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3-F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2a kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2a kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2a kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2a kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF-2a kinase by regulatory RNA was prevented by addition of a high concentration of poly(1) . poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2a kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2a kinase, are consistent with a physiological role for the dsRNA-dependent eIF-2a kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed.Many mRNAs have structural elements in the 5' and 3' untranslated regions which may regulate their overall translational efficiency. Translation of several mRNAs has been shown to involve the interaction of structural elements with specific initiation factors and ribosomes [1 -31, factors which destablize mRNAs 14 -61, factors which mobilize the mRNA from the nucleus to the cytoplasm [7], and factors which regulate the translation of mRNAs in response to cellular needs as has been demonstrated for the iron-responsive elements in the 5' untranslated region of ferritin mRNA [8, 91. Secondary structures within single-stranded viral RNAs have also been shown to regulate the activity of the dsRNA-dependent eIF-2a kinase, a potent inhibitor of protein synthesis [lo-121. The findings that eIF-2a becomes pho...

Involvement of Double-stranded RNA-activated Protein Kinase in the Synergistic Activation of Nuclear Factor-κB by Tumor Necrosis Factor-α and γ-Interferon in Preneuronal Cells

“…However, PKR has also been implicated in controlling cell growth, cell differentiation, and tumor supression (for review, see Refs. 29 and 30), and there is evidence that it can become phosphoryl- ated in the absence of viral infection or dsRNA treatment (59). In PC12 and B12 cells, pretreatment with the PKR-specific inhibitor 2-AP completely blocked TNF-␣/IFN-␥-induced NF-B nuclear translocation and reduced B-dependent gene expression by at least 40% (Figs.…”

“…Second, IFN-␥ may lead to an increased activity of PKR, but signals generated by the presence of both cytokines may be required to target the NF-B⅐IB␤ complex. Third, signals generated by cotreatment could induce the synthesis of or change the structure of a cellular dsRNA or other PKR activator, which could then activate PKR (59). Also, there is evidence for endogenous proteins that act as cellular PKR inhibitors (63,64); therefore cotreatment could generate signals that could counteract the inhibitory roles of these proteins.…”

Action of spontaneously produced beta interferon in differentiation of embryonal carcinoma cells through an autoinduction mechanism.

“…Exogenously added IFN has been shown to have profound and varied effects on differentiation in a number of systems (for a review, see reference 64). Autocrine IFN has been shown to control differentiation in some systems (9,34,43,58,61 to inducers (2,10). On the other hand, no direct evidence for a role for autocrine IFN in the differentiation of pluripotent cells has been presented.…”

Translational Control of Viral Gene Expression in Eukaryotes

“…IFNs, produced in response to viral infection, induce the transcriptional activation of PKR, resulting in a high level of PKR expression. During virus infection, the binding of virus-encoded dsRNA to PKR initiates the PKR activation process, which includes the aforementioned dimerization and autophosphorylation events (84,301,373). Once activated, PKR phosphorylates eIF2␣ to limit mRNA translation.…”