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-ribofuranosylbenzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.