CHARACTERIZATION AND MAPPING OF THE DOUBLE-STRANDED REGIONS INVOLVED IN ACTIVATION OF PKR WITHIN A CELLULAR RNA FROM 3T3-F442A CELLS

PKR is a doubled-stranded RNA-dependent protein kinase which is implic ated in the regulation of several cellular processes, including cell p roliferation. PKR undergoes phosphorylation and activation in mouse em bryonic 3T3-F442A cells in response to endogenous RNA(s), Activation o f PKR is related to growth and differentiation of these cells. A cellu lar regulatory RNA (R-RNA) which activates PKR has been isolated from these cells and its cDNA partially sequenced. Here we have characteriz ed the R-RNA transcript with respect to nuclease sensitivity and the e xtent of double-stranded structure involved in activation of PKR. The location of the activating sequence was mapped to a contiguous 226/252 nt region of the R-RNA transcript by hybridization to its cDNA fragme nts. Hybridization with a panel of short oligodeoxynucleotides complem entary to the R-RNA, coupled with protein kinase analysis, was used to probe the 252 nt region for critical sequences, Three short non-conti guous sequences which appear most important for activation of PKR were identified within the 252 nt region, Thus, these studies have identif ied specific sequences most important for activation of PKR, Furthermo re, since the above antisense oligodeoxynucleotides inhibit enzyme act ivation, our results exemplify an unusual mode of action of antisense sequences on the activation of PKR by disruption of RNA secondary stru cture.