In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin.
Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.
Nguyen
V T
VT
Laboratoire de Génétique Moléculaire, Ecole Normale Superieure, Paris.
Giannoni
F
F
Dubois
M F
MF
Seo
S J
SJ
Vigneron
M
M
Kédinger
C
C
Bensaude
O
O
D000546:Amanitins / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D000546:Amanitins; D004791:Enzyme Inhibitors; D007364:Intercalating Agents; D011994:Recombinant Proteins; D003609:Dactinomycin; D012319:RNA Polymerase II