Ra. Petryshyn et al., CHARACTERIZATION AND MAPPING OF THE DOUBLE-STRANDED REGIONS INVOLVED IN ACTIVATION OF PKR WITHIN A CELLULAR RNA FROM 3T3-F442A CELLS, Nucleic acids research, 25(13), 1997, pp. 2672-2678
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.