Nucleic Acids Research, Vol 24, Issue 15 2924-2929, Copyright © 1996 by Oxford University Press
VT Nguyen, F Giannoni, MF Dubois, SJ Seo, M Vigneron, C Kedinger and O Bensaude
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.
ARTICLES
In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin
Laboratoire de Genetique Moleculaire, Ecole Normale Superieure, Paris.
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