Rtr1 Is a CTD Phosphatase that Regulates RNA Polymerase II during the Transition from Serine 5 to Serine 2 Phosphorylation

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1 Molecular Cell, Volume 34 Supplemental Data Rtr1 Is a CTD Phosphatase that Regulates RNA Polymerase II during the Transition from Serine 5 to Serine 2 Phosphorylation Amber L. Mosley, Samantha G. Pattenden, Michael Carey, Swaminathan Venkatesh, Joshua M. Gilmore, Laurence Florens, Jerry L. Workman, and Michael P. Washburn Supplemental Experimental Procedures TAP Purification from Chromatin-Solubilized Extracts TAP-tagged yeast strains were grown in 1X YPD to OD 600 = Cells were lysed as previously described in TAP extraction buffer (40mM Hepes, ph 7.5, 350mM NaCl, 0.1% Tween-20, 0.5mM DTT, and 1X protease inhibitor cocktail (Roche, complete EDTA-free tablets) (Krogan et al., 2002; Puig et al., 2001; Rigaut et al., 1999). Following lysis, cells were treated with 120ug of heparin (sodium salt, Sigma) and 100 units of DNase I (amplification grade, Sigma) for 10 minutes at room temperature. The lysates were then clarified by centrifugation prior to the addition of 250uL of a 50% slurry of IgG-Sepharose (GE Life Sciences) and incubation overnight at 4 C. Protein complexes were released from the IgG- Sepharose by incubation with 10uL of AcTEV protease (Invitrogen) for one hour at 30 C. The eluate was then diluted in CaM binding buffer (10mM Tris, ph 8.0, 1mM magnesium acetate, 1mM imidazole, 2mM CaCl 2, 0.1% NP-40, 300mM NaCl, 1X protease inhibitors) prior to incubation with 250uL of a 50% slurry of CaM-Sepharose (GE Life Sciences). Proteins were eluted with CaM binding buffer containing 2mM EGTA rather than CaCl 2.

2 MudPIT Mass Spectrometry In order to analyze the intact mixture of purified protein complexes, TCA-precipitation and MudPIT analyses were performed as follows. The TCA-precipitated proteins were resuspended in 8M Urea followed by reduction and alkylation prior to digestion with endoproteinase LysC (Roche) for 6 hours at 37 C. The solution was then adjusted to 2M urea prior to the addition of CaCl 2 and trypsin (Promega). The reactions were quenched with 1% formic acid and then the digested peptide mixtures were applied to a split- microcapillary column as previously described (Florens et al., 2006). A 12-step MudPIT run was performed on each sample, where peptides were eluted into an LTQ ion trap mass spectrometer equipped with a nano-liquid chromatography electrospray ionization source (ThermoFisher) (Florens et al., 2006). SEQUEST was employed to match tandem mass spectra to amino acid sequences consisting of 5879 S. cerevisiae protein sequences downloaded from the National Center for Biotechnology ( release) and 178 contaminants proteins including G. gallus calmodulin, used as a control for contamination from the CaM Sepharose beads used in the TAP purification. The database also included randomized versions of each non-redundant protein entry to estimate false discovery rates (FDR). The peptide false discovery rates (FDRs) are given in Table S5 for each of the protein preparations shown in Figure 1. The FDR value for each TAP was kept at less than 2%. The protein sequences for ubiquitin (Ubi4) were preprocessed in order to reflect the mature, processed form of ubiquitin expressed in the cell. All SEQUEST searches were performed with a static modification of +57 daltons added to cysteine residues to account for carboxamidomethylation. In order to identify oxidized methionines we performed dynamic searches of +16 daltons. Spectra/peptide matches were initially filtered using DTASelect/CONTRAST followed by manual validation to ensure that

3 analogous peptides were not assigned to multiple proteins. The spectral abundance factor (SAF) was calculated as described in Florens et al. (2006). Construction of the HFH-Tagging Vector paj4 The starting vector pag60, obtained from the European Saccharomyces Cerevisiae Archive for Functional Analysis (EUROSCARF), contains the Candida albicans URA3 gene and transcriptional control sequences from the A. gossypii translation elongation factor 1 alpha (TEF) gene (Goldstein et al., 1999). The pag60 vector was first modified by inserting a sequence encoding for a 6x His, 3x FLAG epitope and TEV protease recognition site. The DNA sequence was digested with SalI and BamHI and ligated into SalI/BamHI-digested pag60 resulting in the vector paj1. The 3x HA epitope and ADH terminator sequence was PCR amplified from pfa6a-ha-kanmx6 which has been described by Longtine et al (Longtine et al., 1998). The PCR fragment was then inserted into paj1 following the TEV protease recognition site, the sequence encoding a 3x HA epitope and ADH terminator was inserted between XmaI and BglII sites. Construction of the Rtr1-His 6 -HA 1 -Protein A Bacterial Expression Vector Rtr1-His 6 -HA 1 -Protein A sequence was cloned from the BG1805 vector containing the Rtr1 sequence, which was obtained from Open Biosystems (Gelperin et al., 2005). The clone was PCR amplified using primers directed against the ATG of Rtr1 containing a 3 CACC overhang to enable the TOPO cloning reaction (5 -CACC ATG GCG ACG ATT GAA GAT AT-3 ) and the stop codon of the Protein A ZZ domain (5 -CTC GAG TCA CTG ATG ATT CGC GTC TAC-3 ) from the BG1805 vector (Gelperin et al., 2005). The PCR product was

4 then cloned into the pentr / D-TOPO vector using a pentr Directional TOPO Cloning Kit (Invitrogen). Following clone verification by sequencing, LR Clonase (Invitrogen) was used for recombination reactions with the Gateway destination vector pet- DEST42. The resulting clones were verified by sequencing and then transformed into BL21 DE3 plyss cells (Stratagene) for induction of protein expression. Rtr1-His 6 -HA 1 -Protein A Recombinant Protein Two-Step Affinity Purification Following induction of Rtr1 expression with Isopropyl β-d-1-thiogalactopyranoside, BL21 DE3 plyss cells (Stratagene) were lysed in TAP extraction buffer by sonication and the resulting extract was clarified through centrifugation. The lysate was then incubated with 1 ml of a 50% slurry of IgG-Sepharose and incubated overnight at 4 C. Protein complexes were released from IgG-Sepharose by incubation with 10 μl of PreScission Protease (GE Life Sciences) in 3C protease buffer (50mM Tris-HCl ph 8.0, 100mM NaCl, 1mM EDTA, 1mM DTT) overnight at 4 C, which cleaved between the HA and Protein A tags in the recombinant protein. After cleavage, the column was washed with TAP extraction buffer (lacking DTT) prior to addition of 500μL of Anti-HA resin (Sigma) and incubated for 3 hours at 4 C. Purified Rtr1 was eluted with 100μg/mL HA peptide (Sigma) in TAP buffer (without DTT). Chromatin Immunoprecipitation Assays For the H14 and H5 IgM monoclonal antibodies, an alternative wash buffer was used as described by Ahn et al., To facilitate antibody/protein G interactions, 2uL of Anti- IgM/IgG (Sigma) was added as previously described (Komarnitsky et al., 2000). Additional antibodies used in these studies are listed in Table S2. The input and immunoprecipitated

5 DNA were analyzed by qpcr using a Biorad icycler and SYBR Green master mix (Stratagene). To determine the approximate concentration of each sample, a standard curve was generated for each set of PCR reactions using serial dilutions of yeast genomic DNA in duplicate. The percent enrichment was calculated by dividing the percent IP of the experimental sample by the percent IP of a control PCR reaction using a region of chromosome 5 that RNAPII does not bind (Komarnitsky et al., 2000). The sequences for the PCR primers are given in Table S3. Construction of the Rtr1-TAP and Rtr1-C73A-TAP Expression Vectors and Strains A RTR1 fragment (-266 to +678) was amplified from BY4741 yeast genomic DNA and cloned into the Nco I site of pbs1539 (Puig et al., 2001) to create RTR1-TAP. Mutant plasmids were created by replacing the BstXI and BsmI wild type RTR1 fragment from prtr1-tap with the corresponding mutant sequence from RTR1-C73A-pEXP5. The resulting plasmids were verified by sequencing. Yeast strains were created by transforming BY4741 with PCR product amplified from wild type or mutant RTR1-TAP plasmids and selecting transformants on complete synthetic media lacking uracil. Copper Induction Experiments Using p416-cup1-driven URA3 Plasmids For strain construction, the p416-cup1 plasmids containing either WT-Rtr1-HA or C73S- Rtr1-HA were transformed into rtr1 cells. Cells were grown to an OD 600 =0.5 in complete synthetic media lacking uracil prior to induction. An aliquot of cells was taken before the addition of 100µM copper sulfate to serve as the zero time point. Further aliquots were taken at the indicated time points following copper addition. Cells from each time point were then

6 used for protein extraction followed by western blot analysis.

7 Supplemental Figures Figure S1. Gel Shift Analysis of Increasing Concentrations of Rpb3-TAP- and Rtr1-TAP- Purified RNAPII The 32 P-labeled free DNA and shifted DNA:RNAPII complexes are indicated to the right of the figure. Quantitation of the DNA:RNAPII complexes was performed with ImageQuant with the percent of maximum value for each lane given at the bottom of the panel.

8 Figure S2. Normalization of Rtr1-HFH Enrichment to Rpb3 Does Not Effect the Location of the Rtr1 Peak The data normalized to Rpb3 enrichment is shown in gray squares. The data obtained from the Rtr1-HFH enrichment prior to the additional normalization is shown in black squares for comparison purposes.

9 Figure S3. Quantitation of the Fold Enrichment of S2-P RNAPII Occupancy across the PMA1 (A) and PYK1 (B) Open Reading Frames Using the H5 Monoclonal Antibody Data is expressed as average fold enrichment ± standard deviation (n=3), which was calculated by dividing the percent IP of the indicated regions over the percent IP of a control region on chromosome 5 where RNAPII does not bind.

10 Figure S4. SDS-PAGE Analysis of Rtr1-HA 1 -His 6 Protein Purity following Two-Step Affinity Purification Protein was visualized by silver staining (Silver) or western blotting with anti-ha (WB).

11 Figure S5. Rtr1 Can Dephosphorylate High-Mobility Forms of Rpb1 Containing S2-P Quantitation of three replicate phosphatase reactions performed on CTDK-I modified GST- CTD. Quantitation was performed using ImageQuant on western blots performed using the H5 monoclonal antibody that detects serine 2 phosphorylation and is expressed as the average percent maximum intensity ± standard deviations. Quantitation was performed on the slower migrating bands seen in Figure 6B.

12 Figure S6. Induction of WT-Rtr1 but Not C73S-Rtr1 Results in Dephosphorylation of S5- P RNAPII A. Western blot analysis of whole cell extracts from the indicated strains. Copper was added the cultures for the indicated time and aliquots were taken and used for extract preparation. Antibodies used for detection are indicated to the right. B. Quantitation of three replicate western blots as shown in A. Data is expressed as average level of S5-P RNAPII normalized

13 to the amount of Pgk1 for loading and then compared to the same measurement at time 0 ± standard deviations (n=3). Supplemental Tables Table S1. Yeast Strains Used in This Study Name Genotype Source Rpb8-TAP MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rpb8-TAP::HIS3MX6 Open Biosystems Rpb3-TAP MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rpb3-TAP::HIS3MX6 Open Biosystems Rpb7-TAP MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rpb7-TAP::HIS3MX6 Open Biosystems Rpb11-TAP MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rpb11-TAP::HIS3MX6 Open Biosystems yam28 MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rpb3-TAP::HIS3MX6 Rtr1-His6-FLAG3-HA3::URA3 rtr1 Δ MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 rtr1::kanmx6 Open Biosystems yam25 MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rtr1-His6-FLAG3-HA3::URA3 YSP193 MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rtr1-TAP::URA3 YSP194 MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Rtr1-C73A-TAP::URA3 yam33 MATa his3-1 leu2-0 met1-50 ura3-0 rtr1::kanmx6 [p416cup1-ura3-rtr1-ha], Gibney et al., 2008 yam34 MATa his3-1 leu2-0 met1-50 ura3-0 rtr1::kanmx6 [p416cup1-ura3-rtr1-c73s-ha], Gibney et al., 2008 Table S2. Antibodies Used in This Study Antibody Name Supplier Applications 8WG16 (UM CTD) Covance Western blot H14 (S5-P CTD) Covance Western blot, H5 (S2-P CTD) Covance Western blot, anti-tap Open Biosystems Western blot anti-flag M2-peroxidase Sigma Western blot anti-ha-peroxidase (12CA5) Roche Western blot anti-ha (3F10, high affinity) Roche anti-rpb3 (goat polyclonal) Santa Cruz Biotechnologies Western blot, anti-rpb3 (1Y26, monoclonal) Neoclone Western blot anti-pgk1 Invitrogen Western blot Peroxidase anti-peroxidase (produced in rabbit) Sigma Western blot

14 Table S3. SAF Values for Proteins Plotted in Figure 1C Fraction 38 Fraction 40 Fraction 42 Fraction44 Fraction 46 SAF SAF SAF SAF SAF RPB RPB RPB RPB RPB RPB RPB RPB RPB RPB RPB RPB SPT SPT TFG RTR

15 Table S4. Primers Sequences Used for, Northern Blot Probe Preparation, and qrt- PCR Name Primer sequence (5' to 3') Used for Reference Control F (Chro V) PMA1-73 PMA1 +72 PMA PMA PMA PMA PMA PMA PYK1-257 PYK1-142 PYK1 +32 PYK PYK PYK PYK PYK PYK SCR1-UP GGC TGT CAG AAT ATG GGG CCG TAG TA CCC CAG CTA GTT AAA GAA AAT CAT TGA A AAA GCC TGC TAA GAC TTA CGA TGA CG CGA CGA CGA AGA CAG TGA TAA CG AAG TCG TCC CAG GTG ATA TTT TGC A GTT TGC CAG CTG TCG TTA CCA CCA C CTA TTA TTG ATG CTT TGA AGA CCT CCA G GAT ACA CTA AAA AGA ATT AGG AGC CAA C GAA AAT ATT TGG TAT CTT TGC AAG ATG GAA GAC ACT AAA GGT ACC TAG CAT CAT ATG CAT GGT CCC CTT TCA AAG TTA CGT TGT TGC TGG TTC TGA CTT G ACA ACG CCA GAA AGT CCG AAG A AGA TCA TGT ACG TTG ACT AC ACC ATC AGA GAA GTC TTG GGT GCT ATC TTG GAT GGT GCT GAC CTT GGT TAC CAG ATG CCC AAG A GAC ATG GTT TTT CTT TTC AAC TC TGA TCA ACT TAG CCA GGA CAT CCA Control R (Chro V) PMA1 +71 PMA PMA PMA PMA PMA PMA PMA PYK1-327 PYK1-23 PYK PYK PYK PYK PYK PYK PYK SCR1-DOWN CAC CCC GAA GCT GCT TTC ACA ATA C TCT TGA GTT GGC TGA TGA GCT GAA TTC TTC TGG AAC TGG TCT AGC TTC AC ATT GAA TTG GAC CGA CGA AAA ACA TAA C AAC GAA AGT GTT GTC ACC GGT AGC TTC TTC TTT CTG GAA GCA GCC AAA C TGC CCA AAA TAA TAG ACA TAC CCC ATA A CAA GAA AGA AAA AGT ACC ATC CAG AG GTA AAT TTG TAT ACG TTC ATG TAA GTG GAA TGC TTG TGA TGT CTT CCA AGT TGA TTG GTG TCT TGT AAA TAG AAA CA CAA TGA CAG ACT TGT GGT ATT CG CGT CAC AAG CCT TAG CGT ACT TG AGA TCT TAC CGG CGT TCA AAG CTG GGA TTT CAA TAC CCA AGT TTC TCA TGT CAT CGT AGT TTG AAG ATA CCG AAT TCC TTA GCC GCA ACA CCT CAT CGT TAT GAC G GAT CAA CTT GCA CAA TTA TCC GA, qrt-pcr, qrt-pcr Northern Komarnitsky et. al, 2000 Komarnitsky et. al, 2000 Komarnitsky et. al, 2000 Kim et. al, 2004 Komarnitsky et. al, 2000 Bucheli et. al, 2005 Bucheli et. al, 2005 Brown et. al, 2001 NRD TGG TAG CAG ACG TGA ACG TGA ACG TGA AAG NRD TTA GCT TTG TTG TTG TTG CTG CTG CTG TTG Northern MRPL17 +1 ATG AAG GTA AAT TTA ATG TTG AAA AGA GGG MRPL TCC GCT AAA AGA AAT TCT GTT TTG TTA AAG Northern PMA GTT TGC CAG CTG TCG TTA CCA CCA C Kim et. al, 2004; Northern PMA TGC CCA AAA TAA TAG ACA TAC CCC ATA A Komarnitsky et. al, 2000 o-act1-2 TTG GGT TTG GAA TCT GCC GG o-act1-1 GTG AAC GAT GAA TGG ACC AC qrt-pcr SCR1 QPCR F AGG CTG TAA TGG CTT TCT GGT GGG A SCR1 QPCR R ATA TGT GCT ATC CCG GCC GCC TCC A qrt-pcr

16 Table S5. False Discovery Rates for the RNAP Preparations TAP bait Peptide FDR (%) Rpb Rpb Rpb Rpb Rtr Mock (BY4741) Supplemental References Ahn, S.H., Kim, M., and Buratowski, S. (2004). Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. Mol. Cell 13, Brown, C.E., Howe, L., Sousa, K., Alley, S.C., Carrozza, M.J., Tan, S., and Workman J.L. (2001). Recruitment of HAT Complexes by Direct Activator Interactions with the ATM- Related Tra1 Subunit. Science 292, Bucheli, M.E., and Buratowski, S. (2005). Npl3 is an antagonist of mrna 3' end formation by RNA polymerase II. EMBO J. 24, Florens, L., Carozza, M.J., Swanson, S.K., Fournier, M., Coleman, M.K., Workman, J.L., and Washburn, M.P. (2006). Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors. Methods 40, Gibney, P.A., Fries, T., Bailer, S.M., and Morano, K.A. (2008). Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins. Euk. Cell 7,

17 Gelperin, D.M., White, M.A., Wilkinson, M.L., Kon, Y., Kung, L.A., Wise, K.J., Lopez-Hoyo, N., Jiang, L., Piccirillo, S., Yu, H., et al. (2005). Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 19, Goldstein, A.L., Pan, X., and McCusker, J.H. (1999). Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae. Yeast 15, Kim, M., Ahn, S.H., Krogan, N.J., Greenblatt, J.F., and Buratowski, S. (2004). Transitions in RNA polymerase II elongation complexes at the 3' ends of genes. EMBO J. 23, Komarnitsky, P., Cho, E.J., and Buratowski, S. (2000). Different phosphorylated forms of RNA polymerase II and associated mrna processing factors during transcription. Genes Dev. 14, Krogan, N.J., Kim, M., Ahn, S.H., Zhong, G., Kobor, M.S., Cagney, G., Emili, A., Shilatifard, A., Buratowski, S., and Greenblatt, J.F. (2002). RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22, Longtine, M.S., McKenzie, A., 3rd, Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J.R. (1998). Additional modules for versatile and economical PCRbased gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotech. 17,

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