A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR

Abstract

We report a role for the 3-untranslated region in control of mRNA splicing and show that human TNF-␣ 3 UTR harbors a cis-acting element that renders splicing of precursor transcripts dependent on activation of PKR, the RNA-activated protein kinase that phosphorylates eukaryotic initiation factor 2 (eIF2). When this element, designated 2-APRE, is present, splicing becomes sensitive to inhibition by the PKR inhibitor, 2-aminopurine, or by coexpression of transdominant-negative mutant PKR. Our results reveal that activation of PKR is required for splicing of mRNA when precursor transcripts contain the 2-APRE and that increased expression of wild-type PKR enhances their splicing efficiency. Thus, PKR responds as trans-acting factor to the 2-APRE. 2-APRE RNA forms a stable, 17-bp stem-loop structure and strongly activates PKR in vitro, inducing eIF2␣ phosphorylation. Despite its ability to activate PKR during splicing, the 2-APRE within the 3 UTR does not affect translation efficiency of the resulting TNF-␣ mRNA in transfected cells. PKR and the 3 UTR thus interact during mRNA splicing to confer a novel type of regulation on expression of the TNF-␣ gene.

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions

“…After transcription, pre-mRNA is processed through a series of RNA splicing events. In a most recent study, an element in the 3'-UTR of tumor necrosis factor a mRNA was found to render this RNA processing mechanism (Osman et al, 1999). After that, mRNAs are to be exported out from the nucleus through the nuclear pore.…”

Protein kinase inhibitor 2-aminopurine overrides multiple genotoxic stress-induced cellular pathways to promote cell survival

“…To determine whether 2-AP can interfere with the stress-induced stabilization of p53, we pretreated wild-type (wt) p53-containing NIH 3T3 cells with 10 mm 2-AP or solvent only for 2 h and then exposed the cells to AD, UV, or g-radiation for various lengths of time. This concentration of 2-AP was shown to induce its observed cellular effects (Andreassen and Margolis, 1992;Jarrous et al, 1996;Strong et al, 1998;Osman et al, 1999). Within 1 h after addition of AD in the absence of 2-AP, the protein level of p53 increased when compared to that of the cells without AD treatment (Figure 1a).…”

“…PKR is a Ser/Thr kinase expressed constitutively at low levels in most cells but is induced by viruses, dsRNA, and IFN (Kaufman, 2000). 2-AP prevents the establishment of the antiviral state in IFNtreated cells (Wathelet et al, 1989) and has been instrumental in several functional studies of PKR, such as elucidating the ability of PKR to regulate the expression of tumor necrosis factor-a gene (Jarrous et al, 1996;Osman et al, 1999), the activation of transcription factor NF-kB (Cheshire et al, 1999), or explaining the role of PKR in reovirus-mediated oncolysis of tumor cells (Strong et al, 1998). Our previous studies correlated the absence of wt PKR function with downregulation of two p53 downstream targets, p21 and mdm2 genes and a partial reduction of stress-induced p53 phosphorylation on serine 18 (Cuddihy et al, 1999a).…”

“…The final 2-AP treatment concentration was 2-10 mm as indicated (Andreassen and Margolis, 1992;Jarrous et al, 1996;Strong et al, 1998;Osman et al, 1999;Dimitrova and Gilbert, 2000). Non-2-AP-treated cells were incubated with sterile NaOH at final concentrations of 0.5-2.5 mm as control to ensure that they were exposed to the same amount of NaOH as the 2-AP-treated cells.…”

“…2-AP has been extensively used as a selective and potent inhibitor of PKR (Hu and Conway, 1993;Jarrous et al, 1996;Osman et al, 1999;Ben-Asouli et al, 2002). PKR is a Ser/Thr kinase expressed constitutively at low levels in most cells but is induced by viruses, dsRNA, and IFN (Kaufman, 2000).…”