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

Abstract

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.

Transcriptional Blockade Induces p53-dependent Apoptosis Associated with Translocation of p53 to Mitochondria

“…␣-Amanitin binds directly to the LS of pol II (26) and specifically induces its ubiquitination and consequent degradation (27). Consistent with previous observations (28), cells treated with ␣-amanitin manifested a time-dependent decrease in the total amount of mRNA and were unable to resume mRNA synthesis.…”

“…␣-Amanitin Induces pol II Degradation and p53 Accumulation without DNA Double-strand Breaks-␣-Amanitin induces the ubiquitination of the C-terminal domain of the LS of pol II (29) and consequent proteasome-mediated protein degradation (27). We examined the effect of ␣-amanitin on the abundance of the LS of pol II in p53 ϩ/ϩ or p53 Ϫ/Ϫ HCT116 cells by immunoblot ( Fig.…”

Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

“…Rabbit polyclonal anti‐GFP (Abcam, ab290; dilution 1:2,000); mouse monoclonal anti‐alpha‐Tubulin (Sigma‐Aldrich, T9026; dilution 1:1,000); rabbit polyclonal anti‐ASH2L (gift from Winship Herr; dilution 1:2,000); rabbit polyclonal anti‐Lamin A (Sigma‐Aldrich, L1293; dilution 1:2,000); mouse monoclonal anti‐RPB1 CTD (1PB 7C2, (Nguyen et al , 1996; dilution 1:20,000); rat monoclonal anti‐RPB1 CTD phospho Ser2 (Active Motif, 61083; dilution 1:2,000); rabbit polyclonal anti‐H3K4me3 (Abcam, ab8580; dilution 1:1,000); rabbit polyclonal anti‐H3K36me3 (Abcam, ab9050; dilution 1:1,000); rabbit polyclonal anti‐H3K9ac (Abcam, ab4441; dilution 1:1,000); rabbit monoclonal anti‐H2Bub (Cell Signaling Technology, 5546; dilution 1:5,000); rabbit polyclonal anti‐H3 (Abcam, ab1791; dilution 1:2,000), mouse monoclonal anti‐GCN5 2GC 2C11 (dilution 1:1,000; Brand et al , 2001) or 5GC 2AG (dilution 1:1,000; Krebs et al , 2010); rabbit polyclonal anti‐ZZZ3 2616 and anti‐mADA3 2678 (dilution 1:1,000; Nagy et al , 2010); rabbit polyclonal anti‐SPT20 3006 (dilution 1:1,000; Krebs et al , 2011); mouse monoclonal anti‐TRRAP 2TRR 2D5 dilution (1:1,000; Robert et al , 2006), rabbit polyclonal anti‐SGF29 2461 dilution (1:1,000) and rabbit polyclonal anti‐YEATS2 2783 (dilution 1:1,000; Nagy et al , 2010). …”

Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro

“…Interestingly, Pol II undergoes degradation in cells treated with ␣-amanitin, an inhibitor of its transcription (13). This amatoxin binds specifically to RNA Pol II (14,15), and it arrests the elongating polymerase in a conformation that inhibits incorporation of a subsequent nucleoside triphosphate to the nascent RNA transcript (16,17).…”