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. 2019 Nov 29;9(1):17914.
doi: 10.1038/s41598-019-54027-y.

A SUMO-dependent pathway controls elongating RNA Polymerase II upon UV-induced damage

Irina Heckmann  1 , Maximilian J Kern  1 , Boris Pfander  2 , Stefan Jentsch  1
Affiliations

A SUMO-dependent pathway controls elongating RNA Polymerase II upon UV-induced damage

Irina Heckmann et al. Sci Rep. .

Abstract

RNA polymerase II (RNAPII) is the workhorse of eukaryotic transcription and produces messenger RNAs and small nuclear RNAs. Stalling of RNAPII caused by transcription obstacles such as DNA damage threatens functional gene expression and is linked to transcription-coupled DNA repair. To restore transcription, persistently stalled RNAPII can be disassembled and removed from chromatin. This process involves several ubiquitin ligases that have been implicated in RNAPII ubiquitylation and proteasomal degradation. Transcription by RNAPII is heavily controlled by phosphorylation of the C-terminal domain of its largest subunit Rpb1. Here, we show that the elongating form of Rpb1, marked by S2 phosphorylation, is specifically controlled upon UV-induced DNA damage. Regulation of S2-phosphorylated Rpb1 is mediated by SUMOylation, the SUMO-targeted ubiquitin ligase Slx5-Slx8, the Cdc48 segregase as well as the proteasome. Our data suggest an RNAPII control pathway with striking parallels to known disassembly mechanisms acting on defective RNA polymerase III.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
S2-phosphorylated Rpb1 disappears upon DNA damage induction via a mechanism involving the proteasome and the segregase Cdc48. (A) Levels of Rpb1 and modified forms in WT cells treated with UV irradiation (400 J/m2) followed by a recovery time course in YPD media in the dark, which blocks repair by photolyase. For the western blots (WB) antibodies against the C-terminal domain (CTD) of Rpb1 were used. 3E10 recognizes phosphorylated serine-2 in the CTD,,, 4H8 and 8WG16 recognize the CTD independently of any modification,. Pgk1 served as loading control. (B) Quantification of Rpb1 signals as in (A). Quantification was performed on LI-COR Odyssey with normalization to Pgk1. Data represent mean ± standard deviation calculated from four biological replicates, presented as relative amount to the untreated (-UV) sample. (C + D) Rpb1-S2P levels in WT, proteasome mutant cim3-1 (C) and cdc48-6 or cdc48-3 (D) cells after UV irradiation (400 J/m2) followed by a recovery time course in YPD media. Cells were shifted to 37 °C for 1 h before irradiation and for the recovery phase. For the western blots (WB) anti-Rpb1 (3E10) antibody was used. Dpm1 served as loading control.
Figure 2
Figure 2
Rpb1 of elongating RNAPII is SUMOylated upon UV treatment. (A) Rpb1-S2P and the corresponding SUMO-modified form in WT cells and cells expressing GFP-tagged SUMO after UV irradiation (400 J/m2) followed by a recovery time course in YPD medium. SUMOylation is indicated by mobility shift of Rpb1-S2P with the tagged SUMO protein. The 3E10 antibody was used to detect Rpb1. Dpm1 served as loading control. (B) Immunoprecipitation of Rpb1 with Rpb1-S2P-specific antibody (3E10) from UV-treated and untreated WT, ubc9-1, siz1Δ, siz2Δ and siz1Δ siz2Δ double mutant cells. SUMOylated species of Rpb1 were detected by western blotting (WB) using SUMO-specific antibody.
Figure 3
Figure 3
The SUMO conjugating system is required for UV-induced loss of S2-phosphorylated Rpb1. (A + B) Levels of Rpb1-S2P in WT and ubc9-1 cells (A) and siz1Δ, siz2Δ and siz1Δ siz2Δ double mutant cells (B) after UV irradiation (400 J/m2). In (A) cells were shifted to 37 °C for 1 h before irradiation followed by a recovery in YPD media at 37 °C. The 3E10 antibody was used to detect Rpb1 by western blotting (WB). Dpm1 served as loading control.
Figure 4
Figure 4
The STUbL Slx5-Slx8 regulates the SUMOylated pool of Rpb1. (A) Levels of Rpb1-S2P in WT and slx5Δ slx8Δ cells after UV irradiation (400 J/m2). The 3E10 antibody was used to detect Rpb1 by western blotting (WB). Dpm1 served as loading control. (B) Quantification of Rpb1-S2P levels in WT and STUbL-deficient slx5Δ slx8Δ cells as described in (A). Quantification was performed on a LI-COR Odyssey system with normalization to Pgk1. Shown are mean ± standard deviation calculated from four biological replicates, presented as relative amount compared to the untreated (-UV) sample. (C + D) Immunoprecipitation of Rpb1 with Rpb1-S2P-specific antibodies (3E10) from UV-treated and untreated WT, slx8Δ and slx5Δ slx8Δ cells. Ubiquitylated (C) and SUMOylated (D) species of Rpb1 were detected by western blotting (WB) using ubiquitin- and SUMO-specific antibodies succesively on the same blot. Therfore, control blots for Rpb1 are the same for (C) and (D). (E) Denaturing Ni-NTA pulldowns (Ni-PD) were performed to isolate His-SUMO-conjugates from UV-treated WT and slx5Δ slx8Δ cells following recovery in YPD. SUMOylated species of Rpb1 were detected by western blotting (WB) using 3E10 (Ser2P-CTD) or 8WG16 (CTD) antibodies. SUMOylated Pgk1 served as pulldown control.
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