Prolonged alpha-amanitin treatment of cells for studying mutated polymerases causes degradation of DSIF160 and other proteins

作者：Tsao David C; Park Noh Jin; Nag Anita; Martinson Harold G^*

来源：RNA-A Publication of the RNA Society, 2012, 18(2): 222-229.

DOI：10.1261/rna.030452.111

• alpha-amanitin
• cleavage and polyadenylation
• poly(A)-dependent termination
• RNA polymerase II
• CTD
• DSIF

A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to alpha-amanitin. Mutations of interest are introduced into an alpha-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with alpha-amanitin. Here, we show that cells that are enabled to grow in alpha-amanitin by expression of an alpha-amanitin-resistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an alpha-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in alpha-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of alpha-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in alpha-amanitin, presumably due to the alpha-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the alpha-amanitin-resistant Rpb1-containing polymerase.

• 出版日期2012-2