Dataset Information

Characterization and mapping of the double-stranded regions involved in activation of PKR within a cellular RNA from 3T3-F442A cells.

ABSTRACT: 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.

SUBMITTER: Petryshyn RA

PROVIDER: S-EPMC146784 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Characterization and mapping of the double-stranded regions involved in activation of PKR within a cellular RNA from 3T3-F442A cells.

Petryshyn R A RA Ferrenz A G AG Li J J

Nucleic acids research 19970701 13

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-R ...[more]

PMID: 9185580

Similar Datasets

Project description:1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression.

Project description:BackgroundDouble-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2alpha leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.ResultsHere we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2alpha in yeast.ConclusionConsidering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA and Z-DNA/RNA, and perhaps by altering sensitivity to viral PKR inhibitors. Further implications of our findings for the evolution of the PKR family and for studying PKR/PKZ interactions with viral gene products and their roles in viral infections are discussed.

Dataset Information

Characterization and mapping of the double-stranded regions involved in activation of PKR within a cellular RNA from 3T3-F442A cells.

Publications

Characterization and mapping of the double-stranded regions involved in activation of PKR within a cellular RNA from 3T3-F442A cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets