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 implicated in the regulation of several cellular processes, including cell proliferation. PKR undergoes phosphorylation and activation in mouse embryonic 3T3-F442A cells in response to endogenous RNA(s). Activation of PKR is related to growth and differentiation of these cells. A cellular regulatory RNA (R-RNA) which activates PKR has been isolated from these cells and its cDNA partially sequenced. Here we have characterized the R-RNA transcript with respect to nuclease sensitivity and the extent 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 fragments. Hybridization with a panel of short oligodeoxynucleotides complementary to the R-RNA, coupled with protein kinase analysis, was used to probe the 252 nt region for critical sequences. Three short non-contiguous sequences which appear most important for activation of PKR were identified within the 252 nt region. Thus, these studies have identified specific sequences most important for activation of PKR. Furthermore, since the above antisense oligodeoxynucleotides inhibit enzyme activation, our results exemplify an unusual mode of action of antisense sequences on the activation of PKR by disruption of RNA secondary structure.
Petryshyn
R A
RA
Center for Cancer and Transplantation Biology, Children's National Medical Center, Washington, DC 20010, USA.
Ferrenz
A G
AG
Li
J
J
D016475:3T3 Cells; D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000737:chemistry; D004622:Embryo, Mammalian; D004789:Enzyme Activation; D051379:Mice; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D012313:RNA / Q000737:chemistry*; D011817:Rabbits; D019892:eIF-2 Kinase
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012313:RNA; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase