amanitins and cordycepin

We have analysed the effect of transcription inhibitors on the polysomal localization of 5' terminal oligopyrimidine (TOP-) mRNAs. It is known that, in vertebrates, the translation of this group of mRNAs is regulated according to the growth status of the cell. Mitogenic stimulation of quiescent cells induces a rapid recruitment of TOP mRNAs from translationally inactive light messenger ribonucleoprotein particles to polysomes. It was found that administration of transcription inhibitors to resting cells causes a similar collective translational activation of TOP mRNAs, without affecting global translation. A number of transcription inhibitors were tested in amphibian and mammalian cultured cells. Actinomycin D (act D), cordycepin, and 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole caused a similar activation whereas alpha-amanitin or low doses of act D did not induce the translational response. Concentrations of act D sufficient to induce TOP mRNA translation also induce 40S ribosomal protein S6 kinases 1 (S6K1) activation. Moreover at these concentrations of act D increased phosphorylation of 4E-BP1 was also observed, indicating the involvement of FRAP/mTOR. Consistent with this observation, pretreatment of resting cells with rapamycin suppresses the activation of TOP mRNA translation induced by act D. These results indicate that the effect of act D on translation is mediated by the S6Ks through FRAP/mTOR.

Topics: Amanitins; Animals; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cells, Cultured; Dactinomycin; Deoxyadenosines; Dichlororibofuranosylbenzimidazole; Enzyme Activation; Kidney; Nucleic Acid Synthesis Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Protein Biosynthesis; Protein Kinases; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases; RNA, Messenger; Signal Transduction; TOR Serine-Threonine Kinases; Transcription, Genetic; Vertebrates; Xenopus laevis; Xenopus Proteins

Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS.

Topics: Amanitins; beta-Glucosidase; Chitinases; Dactinomycin; Deoxyadenosines; Glucan 1,3-beta-Glucosidase; Nicotiana; Plants, Toxic; Protein Processing, Post-Translational; Ribosomes; RNA, Plant

Regulation of CYP1A1 gene expression in mouse hepatocytes from C57/BL6 strain in primary culture was investigated with respect to aryl hydrocarbon hydroxylase (AHH) activity and mRNA levels. Small amounts of the CYP1A1 gene transcripts were detected without the presence of any known AHH inducers but after medium change. Maximal level of expression was approximately 6-9 h after the procedure, followed by a decrease to an undetectable level 24 h later. After temporary treatment of hepatocytes for 10 h with cycloheximide, an inhibitor of protein synthesis, AHH activity measured 14 h later was at a normal low level, although medium-change-associated CYP1A1 mRNA were increased by cycloheximide treatment. However, if cells were treated with either actinomycin D, alpha-amanitin or cordycepin, which are inhibitors of RNA synthesis, after exposure to cycloheximide, prominent induction of AHH activity was observed, the levels being almost equal to those for hepatocytes treated with benz[a]anthracene, a potent AHH inducer. With the same experimental protocol benz[a]anthracene-induced AHH activity was enhanced approximately 4-fold. After washing out cycloheximide and/or benz[a]anthracene, the decrease in the mRNA amounts was delayed in the presence of actinomycin D and half amounts were found even 12 h later; while without actinomycin D they reduced with a half-life of 3-4 h. The observations indicate that CYP1A1 gene expression might be regulated at the post-transcriptional level with compensatory mRNA stabilization under conditions of blocked further production, resulting in elevation of AHH activity.

Topics: Amanitins; Animals; Aryl Hydrocarbon Hydroxylases; Benz(a)Anthracenes; Blotting, Northern; Cells, Cultured; Culture Media; Cycloheximide; Cytochrome P-450 Enzyme System; Dactinomycin; Deoxyadenosines; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Enzymologic; In Vitro Techniques; Liver; Mice; Mice, Inbred C57BL; Nucleic Acid Hybridization; Protein Synthesis Inhibitors; RNA Processing, Post-Transcriptional; RNA, Messenger; Time Factors; Transcription, Genetic