Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1

Abstract

Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that—consistent with previous data—DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII.

Ccr4–Not maintains genomic integrity by controlling the ubiquitylation and degradation of arrested RNAPII

“…3A). The ubiquitylation in untreated cells is likely to be caused by DNA damage-independent RNAPII arrest (Somesh et al 2005;Karakasili et al 2014). The amount of polyubiquitylated Rpb1 was significantly reduced in both the treated and untreated not4Δ cells, indicating a Rpb1 ubiquitylation defect.…”

“…The regulation of RNAPII destruction has turned out to be more elaborate than once anticipated. Many factors regulating its removal from genes remain to be identified (Daulny et al 2008;Karakasili et al 2014). Significant unresolved questions include what determines whether RNAPII is salvaged or destroyed and what triggers the recruitment of Rsp5 to initiate the destruction program.…”

Stimulation of RNA Polymerase II ubiquitination and degradation by yeast mRNA 3′-end processing factors is a conserved DNA damage response in eukaryotes

“…The yeast DNA damage-independent pathway uses some components that overlap with DNA damage-dependent degradation, including Def1 and the E2 ubiquitin ligases Ubc4 and Ubc5. In contrast, loss of Rad26, Elc1, and Cul3 did not affect Rpb1 polyubiquitination when Pol II was stalled by a transcription elongation mutation (Karakasili et al, 2014). It is possible that CFIA and CPF contribute to the DNA damage-independent Pol II degradation pathway.…”

“…elongation defects) or nutrient deprivation (e.g. low NTPs) (Karakasili et al, 2014). This degradation pathway appears evolutionarily conserved since Pol II is ubiquitinated and degraded when transcription is arrested by α-amanitin treatment in higher eukaryotes (Lee et al, 2002).…”

“…Pol II can also be stalled independently of DNA damage if the processivity of transcription elongation is reduced by nutrient deprivation or treatment with 6-azauracil (6-AU), a drug that depletes NTP levels. Pol II stalling in the absence of DNA damage likewise leads to Rpb1 ubiquitination and degradation by a distinct yet overlapping pathway (Karakasili et al, 2014). …”