Volume 30, Issue 4, Pages 498-506 (May 2008) Reversal of RNA Polymerase II Ubiquitylation by the Ubiquitin Protease Ubp3  Kristian Kvint, Jay P. Uhler, Michael J. Taschner, Stefan Sigurdsson, Hediye Erdjument-Bromage, Paul Tempst, Jesper Q. Svejstrup  Molecular Cell  Volume 30, Issue 4, Pages 498-506 (May 2008) DOI: 10.1016/j.molcel.2008.04.018 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Cells Lacking UBP3 Activity Are Sensitive to 6-AU (A) Dilution series of yeast cells of the indicated genotype were plated on yeast minimal plates lacking uracil, and containing (6-AU) or not containing (control) 6-AU. Figure S1A further explores the 6-AU sensitivity of ubp3 and ubp12. (B) 6-AU sensitivity of cells lacking BRE5. (C) 6-AU sensitivity of wild-type (WT) or ubp3 cells carrying empty plasmid (pRS426), plasmid expressing wild-type Ubp3 (pRS426-UBP3), or plasmid expressing catalytically inactive Ubp3 (pRS426-ubp3 C469A). Molecular Cell 2008 30, 498-506DOI: (10.1016/j.molcel.2008.04.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Synthetic Genetic Effects Indicating a Role for Ubp3 in RNAPII Transcript Elongation (A) 6-AU sensitivity of cells lacking UBP3 and DST1. (B) Concomitant mutation of UBP3 (or BRE5) and DST1 results in slow growth and sensitivity to elevated temperature. (C) Slow growth and cold sensitivity resulting from concomitant mutation of UBP3 and SPT4. (D) Sensitivity to elevated temperature resulting from concomitant mutation of UBP3 (or BRE5) and SPT4. Molecular Cell 2008 30, 498-506DOI: (10.1016/j.molcel.2008.04.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 ubp3 Mutation Results in RNAPII Hyperubiquitylation and Rapid Degradation in Response to UV Irradiation (A) RNAPII was immunoprecipitated from WT or ubp3 cell extracts, with or without UV irradiation (200 J/m2) of the cells as indicated. Upper panel shows the Rpb1 and Rpb2 bands in the immunoprecipitates (Coomassie-stained PVDF membrane). Lower panel shows the result of subsequent probing of the membrane with anti-ubiquitin antibodies. Identity of proteins is indicated on the right, and size markers are on the left. Please note that more RNAPII was loaded from wild-type than from upb3 cells in this experiment (compare lanes 1 and 2 with 3 and 4, upper panel; see also Figure S3A). (B) Quantification of the results in (A), adjusting for loading. For simplicity of presentation, the level of RNAPII ubiquitylation in wild-type cells in the absence of UV irradiation was set to 1, and the other levels expressed relative to that. (C) Degradation of RNAPII over time in response to a high dose of UV irradiation (300 J/m2 UV light) (at time = 0) in wild-type (WT) and ubp3 cells. The level of RNAPII in cells before UV irradiation was set to 1, and the levels at the indicated times after UV irradiation expressed relative to that. See the Experimental Procedures for details. Error bars indicate standard deviation. Molecular Cell 2008 30, 498-506DOI: (10.1016/j.molcel.2008.04.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Deletion of UBP3 Promotes UV Survival of Repair-Compromised Cells Relative survival (log axis) of cells of the indicated genotype after different levels of UV irradiation is shown (average of seven independent experiments with standard deviation indicated by error bars). Figure S1B further explores the UV sensitivity of ubp3 (and ubp12) strains. Molecular Cell 2008 30, 498-506DOI: (10.1016/j.molcel.2008.04.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Ubp3 Copurifies with RNAPII and Elongation Factors (A) Immunoprecipitation with 12CA5 antibodies from an untagged control strain and a strain expressing HA-tagged Ubp3, respectively. The inputs and precipitates were subjected to western blotting, using anti-Rpb1 antibodies (4H8). (B) Purification of Ubp3 from cells expressing an HA-tagged version of the protein. Purification protocol is outlined on the left. Silver-stained SDS-PAGE gel of the final, highly purified Ubp3 fraction is shown on the right. Identity of constituent proteins (identified by mass spectrometric analysis of individual bands) is indicated on the right, and size markers are on the left. The relevance, if any, of the Ssa2 protein copurifying with Ubp3 is unclear, but the protein has previously been implicated in the DNA damage response through its interaction with the Rad9 checkpoint complex (Gilbert et al., 2003). Molecular Cell 2008 30, 498-506DOI: (10.1016/j.molcel.2008.04.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Ubp3 Can Deubiquitylate RNAPII In Vitro (A) Highly purified RNAPII, ubiquitylated using pure ubiquitylation factors (Somesh et al., 2005), was incubated with the Ubp3 fraction from Figure 6B for the times indicated. The fractions were separated by SDS-PAGE and western blotted with anti-ubiquitin antibodies (Stressgen). Size markers are shown on the left, and the identity of protein band is on the right. (B) Wild-type and mutant Ubp3 complex were incubated with ubiquitylated RNAPII. The ubiquitin chains on RNAPII were not as long as in (A), and the gel was in this case not run as far, explaining the marked difference in appearance between (A) and (B). (C) Tetrameric ubiquitin chains, linked via K48 or K63 as indicated, were incubated with Ubp3 complex as in (A). Strong overexposure of the gel indicated very weak activity (data not shown), but this was insignificant compared to the complete deubiquitylation observed with RNAPII. (D) RNAPII was ubiquitylated as in (A), but with ubiquitin lacking lysines to only allow monoubiquitylation. Wild-type and mutant Ubp3 complex was then incubated with monoubiquitylated RNAPII for the times indicated. The experiments in lanes 10 and 11 contained n-ethyl maleimide (NEM), an inhibitor of cysteine-dependent catalysis (in this case deubiquitylation). Molecular Cell 2008 30, 498-506DOI: (10.1016/j.molcel.2008.04.018) Copyright © 2008 Elsevier Inc. Terms and Conditions