In vivo degradation of RNA polymerase II largest subunit triggered by a-amanitin

α-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 α-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in α-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was α-amanitin dose dependent and was not a consequence of trans-criptional arrest. α-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 α-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated α-amanitin-resistant subunit remained unaffected. Hence, α-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-β-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by α-amanitin cannot be but an irreversible process because of the destruction of RNAPII