Characterization of a cellular RNA that activates PKR in 3T3-F442A cells

Abstract

The double-stranded RNA dependent eIF-2 alpha kinase (PKR) has been implicated in the regulation of a number of cellular processes including cell growth and differentiation. Previous studies using embryonic mouse 3T3-F442A cells have indicated that PKR undergoes phosphorylation and activation in vivo. This activation of PKR has been attributed to a subset of poly(A)(+)-rich cellular RNA (R-RNA) having sufficient secondary structure to interact with the kinase. To characterize the R-RNA activity, a cDNA was prepared which, when transcribed in vitro, gave rise to an RNA transcript that retained its property to activate PKR. The ability of the transcript to activate PKR was sensitive to ribonuclease V1 and was abolished by the addition of high concentrations of poly(I).poly(C). The cloned cDNA was utilized for liquid RNA/DNA hybridization experiments, which disrupted the secondary structure of the R-RNA and for Northern blot analysis. The results from these studies indicated that the measurable RRNA activity was represented in a specific cellular RNA which was responsible for the activation of PKR. Furthermore it was found that the R-RNA was specifically associated with PKR and that this complex could be detected directly in cell extracts. The nucleotide sequence of the cDNA was determined. We propose that this novel cellular RNA may play a critical role in regulating the activation of PKR and thus be an important component in the control of cell growth and differentiation.