Ssu72 Is an RNA Polymerase II CTD Phosphatase Shankarling Krishnamurthy, Xiaoyuan He, Mariela Reyes-Reyes, Claire Moore, Michael Hampsey  Molecular Cell  Volume 14, Issue 3, Pages 387-394 (May 2004) DOI: 10.1016/S1097-2765(04)00235-7

Figure 1 Depletion of Ssu72 and Pta1 Affects RNAP II CTD S5-P Phosphorylation In Vivo (A) Accumulation of RNAP IIO CTD S5-P and depletion of RNAP IIA associated with Ssu72 turnover. Extracts were prepared from the ssu72-td degron strain following incubation at 37°C for the indicated times. Cell extracts were analyzed by Western blot using the indicated antibodies (8WG16, H14, H5, or anti-HA). (B) Plasmid-borne SSU72+ but not the phosphatase-defective ssu72-C15S allele reverses the effect of ssu72-td on CTD S5-P. Western blots were performed with the indicated antibodies; Rpa1 antibody was used as a loading control. Because of the degron tag, a band corresponding to the normal size of Ssu72 is not seen in lanes 1 and 2 with anti-Ssu72 antibody. (C) Overexpression of SSU72+ diminishes CTD S5-P levels, while overexpression of the C15S or D146A phosphatase-defective proteins results in accumulation of CTD S5-P in the wild-type strain W303-1B. (D) Accumulation of CTD S5-P associated with depletion of Pta1. Extracts were prepared from the pta1-td strain following incubation at 37°C for the indicated times. Samples were analyzed by Western blot, as described in (A). Molecular Cell 2004 14, 387-394DOI: (10.1016/S1097-2765(04)00235-7)

Figure 2 Ssu72 CTD Phosphatase Activity Is Specific for S5-P In Vitro (A) Ssu72 catalyzes dephosphorylation of GST-CTDo. The GST-CTDo substrate (150 ng) was phosphorylated at S5 by TFIIH. The indicated amounts of GST-Ssu72, catalytically inactive GST-Ssu72-C15S, or GST alone were added to each reaction. Products were detected by Western blot using the CTD S5-P-specific antibody (H14). The different species of GST-CTDo apparent in (A) and (B) represent CTD with different amounts of phosphate; the two lower mobility forms are substrates for GST-Ssu72. (B) Ssu72 CTD phosphatase activity is inhibited by Na3VO4 but not by NaF. Assays were performed as described in (A) for GST-Ssu72, except that the indicated amounts of phosphatase inhibitors Na3VO4 (lanes 5–7) or NaF (lanes 8–10) were included. Na3VO4 is a specific inhibitor of phosphatases that form phosphocysteine intermediates, whereas NaF is an inhibitor of phosphatases that form phosphoaspartate intermediates (Sharma and Carew, 2002). (C) Ssu72 catalyzes dephosphorylation of GST-CTD S5-P. Assays were performed as described in (A) using 200 ng of GST-CTD and 5 μg of GST-Ssu72. GST-CTD (lane 1, 50% of amount shown in subsequent lanes) was phosphorylated by either TFIIH or Cdc2 kinase, as indicated. Samples were detected by Western blot using antibodies specific for GST, S5-P (H14), or S2-P (H5). GST-CTDo and GST-CTDa denote, respectively, the phosphorylated and unphosphorylated forms of the CTD. The very low-mobility form of GST-CTDo detected by H14 (lane 9) migrates slightly higher than the species detected by H5. (D) Ssu72 catalyzes dephosphorylation of RNAP IIO S5-P. RNAP IIO was phosphorylated with TFIIH and detected with the S5-P-specific antibody (H14). The indicated amounts of recombinant GST-Ssu72 (lanes 2–4), GST-Ssu72-C15S (lanes 5–7), or GST alone (lanes 8–10) were added to each reaction. Molecular Cell 2004 14, 387-394DOI: (10.1016/S1097-2765(04)00235-7)

Figure 3 Ssu72 Phosphatase Activity Is Dispensable for 3′ End Processing In Vitro (A) The phosphatase inhibitors Na3VO4 and NaF have no effect on the coupled cleavage and poly(A) addition of precursor containing the GAL7 poly(A) site. Lane 1, unreacted precursor; lane 2, processing in the absence of phosphatase inhibitors. The position of precursor and cleaved/polyadenylated product is indicated on the right. (B) Defective processing caused by Ssu72 depletion can be rescued by either SSU72 or ssu72-C15S. (C) Loss of Pta1 upon depletion of the degron-tagged Ssu72 is prevented by the presence of either SSU72+ or ssu72-C15S. Samples of the extracts used in the in vitro processing assays in (B) were analyzed by Western blot with the antibodies shown on the righthand side. Molecular Cell 2004 14, 387-394DOI: (10.1016/S1097-2765(04)00235-7)

Figure 4 Depletion of Ssu72 Affects RNAP II Transcription In Vitro (A) Schematic depiction of the double G-less cassette. Transcription initiates at two sites within the G-less I cassette, yielding 110 nt, 130 nt, and 377 nt products following RNase T1 digestion. (B) In vitro transcription reactions were performed using whole-cell extracts from the ssu72-td degron strain that had been incubated at either 25°C or 37°C for 90 min prior to cell lysis. Extracts were incubated at 25°C for 30 min with recombinant GST-Ssu72, catalytically inactive GST-Ssu72-C15S, or buffer alone, as indicated. Following addition of template DNA, transcription complexes were incubated for an additional 30 min at 25°C, followed by addition of α-[32P]-UTP and unlabeled NTP substrates. Samples were processed as described previously (Chen and Hampsey, 2004). Inhibition of transcription by α-amanitin (50 μg/ml) (lane 1) demonstrates that all products (lane 2) are RNAP II specific. Transcription is dramatically impaired using extracts from heat-treated (37°C) cells (lane 3), and this effect is rescued by recombinant GST-Ssu72 (lanes 4 and 5) but not by catalytically defective GST-Ssu72-C15S (lane 6). (C) Western blot analysis of in vitro transcription extracts (B) showing enhanced levels of RNAP IIO S5-P (H14) and diminished levels of RNAP IIA (8WG16) associated with Ssu72 depletion. Antibodies are defined in Figure 1. Molecular Cell 2004 14, 387-394DOI: (10.1016/S1097-2765(04)00235-7)