Examining the Complexity of Human RNA Polymerase Complexes

Article pubs.acs.org/jpr

Examining the Complexity of Human RNA Polymerase Complexes using HaloTag Technology Coupled to Label Free Quantitative Proteomics Danette L. Daniels,*,† Jacqui Méndez,† Amber L. Mosley,‡ Sreenivasa R. Ramisetty,§ Nancy Murphy,† Hélène Benink,† Keith V. Wood,† Marjeta Urh,† and Michael P. Washburn*,§,∥ †

Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202, United States § Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, Missouri 64110, United States ∥ Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, United States ‡

S Supporting Information *

ABSTRACT: Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization. KEYWORDS: quantitative proteomics, protein interactions, RNA polymerases, mass spectrometry, MudPIT, cellular imaging, normalized spectral abundance factor

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INTRODUCTION Cellular function is intricately regulated and controlled by networks of protein interactions and uncovering the mechanisms of action of these networks is critical to understanding not only normal cellular growth, but also human disease.1 Global mapping of protein complexes and characterization of localization patterns are two aspects of proteomics initiatives aimed to further understanding of overall protein function.2 The use of affinity tags for protein pull-downs coupled with significant and recent advances in mass spectrometry have enabled high-throughput protein interaction studies in yeast.3,4 Such studies for human interactions have been initiated, but are increasingly more complex and lag behind other systems due to the total number of possible interactions to be identified and the complexity of the system.5−9 A key consideration for the analysis of protein interaction network analyses is the choice of the affinity tag.10 To facilitate protein interaction network analyses, multifunctional affinity tags are needed where multiple experiments can be carried out with a single construct. For example, protein interactions coupled with protein localization © 2011 American Chemical Society

can greatly accelerate the biological insight into protein function and after generation of antibodies, has been employed using GFP.9 Employing this same principle, yet without the need for antibodies, we apply here the versatile HaloTag technology11 to protein pull-down and cellular imaging experiments. While multiple technologies exist to map protein interactions, each face the challenge to efficiently isolate interactions, particularly transient or weak interactions, while simultaneously minimizing false positives.12,13 The properties of HaloTag fusion protein lend themselves well to being able to address these challenges. The HaloTag protein fusion, derived from a rare bacterial protein, was specifically engineered through both directed and random mutagenesis, to rapidly, specifically, and irreversibly bind to a choloralkane moiety.11 This moiety can be chemically modified to carry different functional groups including solid surfaces or fluorophores, Received: May 17, 2011 Published: December 12, 2011 564

dx.doi.org/10.1021/pr200459c | J. Proteome Res. 2012, 11, 564−575

Journal of Proteome Research

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proteins. Taken together, these data demonstrate the broad utility of HaloTag technology for the study of protein interaction, localization, and function.

yielding various HaloTag ligands, including a sepharose-based resin, HaloLink, for capture of HaloTag protein fusions and their complexes.11,14,15 Given the rapid binding kinetics to the resin, which is typically complete within 15−30 min, a significant time advantage in both isolation and maintenance of complexes can be achieved. The covalent capture allows low abundant complexes to be isolated without diffusion from the resin and also far fewer cells to be used. Finally, to minimize the number of false positives, the lack of an endogenous equivalent of the HaloTag protein combined with the low nonspecific binding properties of HaloLink resin lead to low background capture of spurious interactions. To translate these features into function, the human eukaryotic RNA polymerases, I, II and III were isolated in HaloTag pull-down experiments using a HaloTag fusion of the shared subunit POLR2H,16 and interacting partners along with levels of background were thoroughly analyzed using highly reproducible quantitative proteomics approaches.17 The three core eukaryotic RNA polymerases (RNAPs) I, II, and III are macromolecular complexes with an average mass greater than 500 kDa, and are responsible for synthesis of both coding and noncoding RNAs.18,19 The RNAP II holoenzyme has historically been the most well studied with the greatest number of identified interacting partners,20−22 however all of the RNA polymerases have regulated interactions with other proteins, resulting in temporally distinct complexes with specific functions.19,23 Previous studies of RNA polymerase interactions and localization have utilized antibodies as well as affinity and fluorescent tags, though have been focused on a subunit unique to a particular polymerase.24−28 Recent proteomics analyses of human RNAP II have resulted in the discovery of novel interactions and biology,29,30 but similar studies have yet to be conducted for RNAP I and III. To attempt to simultaneously study and isolate RNAP I, II, and III and their interacting partners, a subunit shared by all three RNAP complexes, POL2RH, was chosen for this study and fused to HaloTag. This subunit, the human homologue of Rpb8 or ABC14.5, seemed an ideal candidate as its position in the yeast RNAP II structure revealed a surface location without a direct involvement in the catalytic active site.31 Rpb8 however is essential for cell viability in yeast,16 likely playing an indispensable structural role and/or recruiting other important regulatory proteins to the RNA polymerase complexes. Here we present quantitative proteomics data showing the successful capture and detection of all protein subunits from RNAP I, II, and III as well as several known and unknown interacting partners in a single pull-down. As levels of protein expression are a concern with any type of affinity tags or transgene expression, a CMV promoter deletion series, spanning approximately a 50-fold range of expression, was used to examine complex capture versus HaloTag-POLR2H cellular expression. Data revealed efficient isolation of all three polymerases could be achieved at near endogenous levels of expression and also that nonspecific or spurious interactions were not observed at high levels of expression. In addition to identification of known RNAP core subunits and interacting proteins, three novel RNA polymerase-associated proteins were discovered and validated in vivo using reciprocal HaloTag pulldowns. Complementary in vivo cellular imaging experiments were performed with the HaloTag fusions to show both proper nuclear localization, colocalization of POLR2H with the large subunit of RNA Polymerase II, POLR2A, and also characterize the localization patterns of the three novel RNAP-associated

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MATERIALS AND METHODS

Cloning, Cell Lines, Transfection, and Western Blot Conditions

Full-length human POLR2H (NM_006232.2) was obtained from Kazusa DNA Research Institute as a pF1K Flexi Vector (catalog number FXC02496), and further subcloned into pFN21A CMV, pFN22K CMVd1, pFN23K CMVd2, and pFN24K CMVd3 HaloTag Flexi Vectors (Promega) using SgfI and PmeI, generating N-terminal HaloTag fusion constructs for each. MLLT11 (DQ894604), hypothetical protein LOC85395 (NP_478070.1), and hypothetical protein LOC90488 (NP_689474.1) were synthesized in vitro containing flanking SgfI and PmeI sites and cloned into Flexi vector pFN21K CMV respectively. The FLAG-POLR2H construct was obtained from GeneCopoeia, which places FLAG on the N-terminus and utilizes the CMV promoter for expression. For the HaloTag experiments, the HaloTag Control Vector (Promega) was used as a negative control. HEK293T cells (ATCC #CRL-11268) were maintained in DMEM supplemented with 10% FBS at 37 °C in an atmosphere of 5% CO2. Cells were transfected using FuGENE HD transfection reagent (Promega) according to manufacturer’s protocols. For the Western blot POLR2H experiments, anti POLR2H antibody (Abnova) was used at a final concentration of 0.2 μg/ml, and anti-tubulin antibody (Abcam) was used at a final concentration of 0.1 μg/ml. For all blots, secondary detection was done with anti-rabbit IgG AP conjugate (Promega) followed by colorimetric detection with Western Blue Stabilized Substrate for AP (Promega) following the manufacturer's recommendations. HaloTag Mammalian Pull-Down Protocol

For these experiments, HEK293T cells (1.2 × 107) were plated in a 15 cm plate. After reaching 70−80% confluency, typically 18−24 h later, cells were transfected with the various HaloTagPOLR2H fusion constructs (experimental samples) or HaloTag control vector (negative control or mock sample). Twenty four hours post-transfection, cells were harvested and frozen at −80 °C until processing. Cells were lysed in standard mammalian lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1%Na deoxycholate, and Protease Inhibitor cocktail (Promega) supplemented with 1.5 mM MgCl2 and 10 mM KCl, incubated on ice for 5 minutes, homogenized using a Dounce glass homogenizer, and then centrifuged at 14000× g to clear the lysate. Resulting lysate supernatant were added directly to HaloLink Resin (Promega) equilibrated in 1× TBS and 0.1% IGEPAL-CA630, and allowed to bind for 15 minutes at 22 °C with rotation. Five washes using 1× TBS were performed and captured complexes were eluted in 50 μL of 8 M Urea 150 mM Tris pH 8.5 prior to MudPIT analysis. Three biological replicates were analyzed from each of the POLR2H cell lines and two biological replicates were analyzed from the MLLT11, LOC85395, and LOC90488 cell lines. FLAG CoIP Protocol

For FLAG coimmunoprecipitation experiments, similar amounts of HEK293T cells (1.2 × 107) were plated in a 15 cm dish. After reaching 70−80% confluency, typically 18−24 h later, cells were transfected with a FLAG-POLR2H fusion construct (experimental sample) or left untransfected (negative 565



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family members were listed with each protein, but were only counted once for calculation of dNSAF values.34 The dNSAF calculations take the consideration of protein length and the relative spectral abundance of proteins across various preparations to prevent the redundancy in spectral assignment.34NSAF7 was used to create the final reports as shown in Supporting Tables 1−5, Supporting Information. Supporting Tables 1 and 3 include the sequence coverage and unique peptides detected for all the identified proteins and also the selection parameters used for spectral counting. Supporting Tables 2 and 4 report dNSAF values and FDRs are reported in Supporting Table 5. FLAG-POLR2H data is provided in Supporting Tables 7 and 8 (Supporting Information). The nonspecific binding proteins were extracted from the data set by comparing the dNSAF value in each of the individual purifications with the dNSAF value from the mock control (HaloTag or FLAG) which were performed in the same cell type as described previously.17

control or mock sample). Twenty-four hours post-transfection, cells were harvested and frozen at −80 °C until processing. CoIP of complexes was performed using the FLAG Tagged Protein Immunoprecipitation Kit (Sigma) following the manufacturer’s protocol with the following customizations: (a) Lysis and wash buffers were supplemented with 1.5 mM MgCl2 and 10 mM KCl, (b) Protease Inhibitor Cocktail (Sigma) was added to the lysis buffer, (c) the optional resin wash with elution buffer to remove unbound ANTI-FLAG antibody was performed, (d) capture was performed for 2 h, and (e) elution was done with 3× FLAG peptide at twice the concentration suggested or 300 ng/μL to improve elution efficiency. Quantitative Proteomics Analysis of Affinity Purified Complexes

As described previously,17 purified protein samples were digested with LysC/Trypsin enzymes and analyzed by LTQ linear ion trap MS equipped with a nano-LC electrospray ionization source (ThermoFisher) coupled with a Quaternary Agilent 1100 series HPLC pump (Agilent Technologies). The samples were loaded onto a split 3-phase column containing 8 cm of 5-μm C18 reverse phase particles (Aqua, Phenomenex) in a 100 μm fused silica microcapillary column followed by 4 cm strong cation exchange resin (Partisphere SCX, Whatman) and 2 cm reverse phase in 250 μm fused silica microcapillary column. The column was placed in line with mass spectrometer and fully automated 12-step MudPIT run was performed as described previously.17 Each full MS scan (from 400 to 1600 m/z range) was followed by five MS/MS events using datadependent acquisition, and the top five intense ions of each MS scan subjected to Collision Induced Dissociation (CID). The MS2 files were extracted from the RAW files using RAWXtract (version 1.0) and searched using SEQUEST (version 27 (revision 9) with no enzyme specificity against a database of 30406 human proteins (from the National Center Biotechnology Information 03−04−2008 release), 163 common contaminants, such as IgGs, keratins, proteolytic enzymes, and their corresponding 30712 redundant proteins (shuffled sequences) resulting from each non redundant entry and contaminant sequences. These randomized redundant protein sequences were used to estimate the false discovery rates (FDRs).32 In all SEQUEST searches cysteine residues were considered fully carboxy methylated with a static modification of +57 Da and oxidized methionine with dynamic modification of +16 Da. A mass tolerance of 3 amu for precursor ions and 0 amu for fragment ions was used. Spectra/peptide matches were filtered using DTASelect/CONTRAST.33 In this data set, spectrum/peptide matches only passed filtering if they were at least 7 amino acids in length and fully tryptic. The DeltCn was required to be at least 0.08, with minimum XCorr value of 1.8 for singly, 2.5 for doubly, and 3.5 for triply charged spectra, and a maximum Sp rank of 10. Proteins that were subset of others were removed using the parsimony option in DTASelect. After analysis of each biological replicate, the peptides hits from multiple runs compared using DTAselect/CONTRAST.33 After merging all runs, proteins that are subset of others were removed using parsimony option in the DTASelect.33 NSAF v7 (an in-house developed software) was used to create the final report on all nonredundant proteins detected across the different runs, estimate FDRs, and calculate their respective distributed Normalized Spectral Abundance Factor (dNSAF) values.34 Peptides matching to multiple protein

Cellular Imaging

U2OS cells were transfected using FuGENE HD (Promega) according to manufacturer’s recommendation. Twenty-four hours post-transfection cells were labeled with 5uM HaloTag TMR ligand (Promega) in complete media (DMEM and 10% FBS) for 15 min at 37 °C and 5% CO2. TMR-containing media was then replaced with fresh complete media twice and cells were placed back at 37 °C and 5% CO2 for 30 min for washing of unbound ligand. Media was then replaced with fixation solution (4% paraformaldehyde/0.2 M Sucrose/1× PBS, pH 7.5) for 10 min at 22 °C, then replaced with 1× PBS containing 0.1% Triton X-100 for 10 min at 22 °C. PBS-Triton was then removed and replaced with 1× PBS and cells were placed at 4 °C for 18 h. PBS was removed and replaced with block solution (1× PBS/2% normal goat serum/0.01% Triton X-100) for one hour at 22 °C, then incubated with 1× PBS/1% normal goat serum/POLR2A CTD primary antibody at 1:200 (AbCam, ab817) for 1 h at 22 °C. Cells were washed with 1× PBS/1% normal goat serum twice for 10 min each at 22 °C. Wash was replaced with 1× PBS/1% normal goat serum/Alexa 488-goat antimouse secondary 1:400 (Invitrogen, A11001) for 30 min at 22 °C. Finally, cells were gently washed twice with 1× PBS/1% normal goat serum for 10 min each at 22 °C and imaged. Images were acquired on an Olympus Fluoview FV500 confocal microscope (Olympus, Center Valley, PA) containing a 37 °C + CO2 environmental chamber (Solent Scientific Ltd., Segensworth, U.K.) using appropriate filter sets. Bioinformatics

To determine the human homologues of the yeast RNAP subunits,35 we performed BLAST searches through NCBI to identify the sequence homologues of the 31 RNAP subunits. Using this approach, we were able to identify homologues for all 31 yeast proteins. Known interactions and complex compositions were also confirmed through literature searching and searches through the Biological General Repository for Interaction Data sets (BioGRID). We were unable to identify the sequence homologue of the RNAPI subunit Rpa14 in our proteomics analyses. BLAST identified the human protein ″ring finger protein 17″ (NP_112567.2) as a potential Rpa14 relative with 30.4% sequence homologue. However, we were unable to detect this protein in our data set leading to the conclusion that there is not an obvious Rpa14 sequence homologue in human cells in agreement with previous reports in the literature.36 Finally, in an attempt to gain some consistency in the 566



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maximal efficiency and minimal nonspecific binding, can be achieved in the time scale of 15−30 min.11,14,15 Gentle washes are performed to remove nonspecific interactions while maintaining complexes. Due to the covalent linkage on the resin, interacting partners can be released by two methods (Figure 1). The first is elution with a denaturant, such as SDS or Urea, and this will release only interacting protein partners, leaving the starting HaloTag fusion protein covalently behind bound to the resin (Figure 1). The second means of elution utilizes TEV protease, which cleaves its recognition sequence contained between the HaloTag and the protein of interest, releasing the protein initially fused to HaloTag and its respective complexes intact and leaving HaloTag behind on the resin (Figure 1). As a control for determining specific versus nonspecific capture, the HaloTag protein is expressed alone and processed through the entire pull-down protocol identically to the experimental sample (Figure 1). Untransfected cells could also be used a control, but would not identify any protein which may nonspecifically interact with the HaloTag protein.

nomenclature we will refer to a number of the RNAP subunits by acronyms that clearly identify them as components of the RNAP complexes. The acronyms used in this publication along with the NCBI database description, some frequently used alternative names in the literature, and the S. cerevisiae sequence homologues are described in Supplementary Table 6, Supporting Information.

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RESULTS

Development of HaloTag Protein Pull-down Method

To isolate proteins using the HaloTag fusion protein, a genetic construct encoding the HaloTag (HT) protein fused either Nor C-terminally to a protein of interest is first generated (Figure

HT-POLR2H Expression and Pull-down

Upon the basis of analysis outlined in the Bioinformatics section of the Methods section, there are 13, 12, and 17 core subunits in human RNAP I, II and III respectively and a Venn diagram depicting these and the overlapping proteins between each polymerase is shown in Figure 2A. To attempt to capture proteins from all three RNA polymerases in a single pull-down, HaloTag was fused on the N-terminus of POLR2H, a subunit shared among all three (Figure 2A). To study the effects of varying expression, HT-POLR2H was expressed from a CMV promoter deletion series, termed High (p21), Medium (p22), Low (p23), and Ultralow (p24), corresponding to progressive deletions of the full length CMV promoter which result in a corresponding decrease of expression level in most eukaryotic cell lines (Figure 2B). The expression levels of transiently transfected HT-POLR2H across the series were analyzed and compared to the endogenous POLR2H in HEK293T cells using an anti-POLR2H antibody with colorimetric detection and show near endogenous levels of HT-POLR2H using the pFN24 vector (Figure 2B). Fluorescent scanning of gels containing HaloTag fusion proteins bound to its fluorescent TMR ligand revealed expression levels of 50×, 16.5×, 3.5×, 1×fold for High, Medium, Low, and Ultralow constructs respectively in relation to the pFN24 construct (data not shown). HaloTag pull-down experiments were performed for all HT-POL2RH constructs using the same starting number, 1.2 × 107, of transiently transfected cells. Interacting partners were eluted using 8 M urea and detected by silver-staining after gel electrophoresis (Figure 2C). As expected total amount of protein captured decreases as expression level of HT-POLR2H decreases (Figure 2C). Similar protein partners captured were observed by elution with SDS or by cleavage from the resin with TEV protease (data not shown). As a control, the HaloTag protein alone was expressed at the Medium expression level and carried through the same pull-down protocol (Figure 2C).

Figure 1. Schematic of the HaloTag pulldown process. A genetic construct encoding the HaloTag (HT) protein fused either N- or Cterminally to a protein of interest is generated and either transiently or stably expressed in mammalian cells, where it incorporates into its respective physiological complexes. After cellular lysis, the HT fusion protein and its complexes are captured within 15−30 min via irreversible binding of HT to a sepharose-based resin, termed HaloLink. Gentle washes are performed to remove nonspecific interactions while maintaining complexes. Due to the covalent linkage on the resin, interacting partners can be released by two methods. The first is elution of interacting protein partners with a denaturant, such as SDS or Urea, and the second utilizes TEV protease, which cleaves its recognition sequence contained between HT and the protein of interest, releasing the protein initially fused to HT and its respective complexes intact, leaving HT still attached to the resin. As a control, HT protein is expressed alone and processed through the entire pulldown protocol identically to the experimental sample.

1). The HaloTag fusion protein is either transiently or stably expressed in mammalian cells, where it incorporates into its respective physiological complexes. Cells are subsequently lysed and HaloTag fusion proteins and their complexes are captured via irreversible binding of HaloTag to a sepharose-based resin, termed HaloLink (Figure 1). This binding, optimized for both

Identification of RNA Polymerase Subunits

Each HT-POLR2H pull-down experiment was performed in biological triplicates and the presence of core RNAP subunits and interacting protein partners were determined using multidimensional protein identification technology and distributed normalized spectral abundance factors (dNSAFs).17,34 567



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Figure 2. Components of human RNA Polymerase complexes, expression profiles of HT-POLR2H proteins, and HaloTag-based complex isolations. (A) Venn diagram showing the individual and overlapping shared components of human RNA Polymerases (RNAP) I, II, and III. As depicted, POLR2H is a shared component among RNAP I, II, and III. (B) Western blot using an anti-POLR2H antibody showing the relative expression in HEK293T cellular lysates of the HT-POLR2H fusion protein transiently expressed from a vector containing the full-length CMV promoter, pFN21, and progressive CMV promoter deletions, pFN22−24. Also designated and visible is the endogenous POLR2H protein. As a loading control, lysates were also probed for endogenous tubulin using an antitubulin antibody. (C) silver stained SDS gel of proteins eluted after the HaloTag complex isolation protocol from HEK293T cells transiently transfected with POLR2H in progressively decreasing expression level vectors, pFN21−24, corresponding to Lanes 1−4, and also the HaloTag alone control, Lane 5. (D) Western blot using an anti-POLR2H antibody showing the relative expression in HEK293T cellular lysates of the FLAG-POLR2H fusion protein transiently expressed from vectors containing a full-length CMV promoter. Also designated and visible is the endogenous POLR2H protein.

The dNSAFs17,34 were calculated and plotted relative to each other across the HT-POLR2H pull-down series (Figure 3). Levels of background protein capture remained constant across the deletion series, suggesting that increased expression levels of the HT-POLR2H does not lead to more false positives or an

increased amount of spurious interactions (Supporting Table 2, Supporting Information). Data across the deletion series and between replicates revealed consistent and reproducible capture of core RNAP I, II, and III subunits (Figure 3). As predicted by Figure 2C, the total number of core subunits identified 568



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Figure 3. Quantitative proteomics analysis of RNAP subunits identified from HT-POLR2H complex isolations. POLR2H subcloned into pFN21A CMV (high expression, black bars), pFN22K CMVd1 (medium expression, red bars), pFN23K CMVd2 (low expression, blue bars), and pFN24K CMVd3 (ultralow expression, pink bars) HaloTag Flexi Vectors and expressed in HEK293T cells and were affinity purified and analyzed using quantitative proteomics approaches. (A) Shown are dNSAF values for the subunits shared between RNAP subunits. (B) Shown are dNSAF values for RNAP I specific subunits. (C) Shown are dNSAF values for RNAP II specific subunits. (D) Shown are dNSAF values for RNAP III specific subunits. All values are avg ± std deviation of three biological replicates.

decreased slightly as expression level decreased, yet even at the Ultralow expression, 90% of the subunits can be identified (Figure 3). Meanwhile, none of the core RNAP subunits were observed in the HaloTag alone control, indicating the low levels of background within the experiment and high degree of specific capture in the experimental sample (Supporting Table 2, Supporting Information). Upon comparison of the proteins identified with our proteomics analysis to the Venn diagram in Figure 2, we found that we reproducibly detected and identified 30 of the 31 human RNAP subunits. The exception was POLR2F, which was only detected and identified in one replicate of POLR2H in the p21 vector (Figure 3A). All other RNAP subunits were found in at least one of three replicates

using more than one CMV expression construct. Notably, we uniquely detected and identified an isoform of POLR3G, which we will refer to as POLR3G-like (also referred to in the literature as RPC32β37). POLR3G and POLR3G-like are two RNAP III isoforms that have isoform-specific functions in cell growth and transformation.37 Comparison of FLAG and HaloTag Systems

As FLAG coimmunoprecipitation (Co-IP) is a commonly used technology for studying intracellular interactions,38,39 a comparison experiment using the same number of cells was performed using POLR2H fused to the FLAG epitope in the same orientation, transiently expressed from a CMV promoter, 569



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making it directly comparable to the High (pFN21) construct from the HT-POLR2H series (Figure 2C-D). A standard FLAG Co-IP was performed and elution of interacting partners was achieved using a competing FLAG peptide. As a control, untransfected cells were used and processed using the same FLAG Co-IP protocol. FLAG-POLR2H coimmunoprecipitation experiments were performed in triplicate and analyzed using MudPIT mass spectrometry (Supporting Tables 7 and 8, Supporting Information). Using the same number of cells for affinity purification, HaloTag-POLR2H from pFN21 HEK293T cells detected and identified 23 RNAP subunits in 3 of 3 replicates, 4 RNAP subunits in 2 of 3 replicates, and 4 RNAP subunits in 1 of 3 replicates (Table 1). In stark contrast, affinity

replicates (Table 1). In this case using the POLR2H protein as a bait, the HaloTag outperformed the FLAG purification. In addition, the three mock FLAG (Supporting Table 8, Supporting Information) purifications detected on average 299 ± 139 proteins while the four mock HaloTag purifications (two from Supporting Table 2 and two from Supporting Table 4, Supporting Information) detected on average 87 ± 18 proteins, less than 1/2 of the proteins from the FLAG purification. Again, in this case, the HaloTag system outperformed the FLAG system by pulling down fewer proteins in control analyses. Our FLAG control data is consistent with previously published studies. In a prior publication using a less sensitive mass spectrometer, the LCQ-Deca, we averaged 116 proteins in 32 total Flag alone negative control runs in HEK293T cells and HeLA cells with several runs having more than 200 proteins detected and identified (see Supplemental Table 2A in ref 5). The nine Flag alone controls from HEK293 cells provided by Ewing et al. and their analysis of human protein interactions averaged 169 proteins, again using the less sensitive LCQ-Deca technology.6 A recent paper from the Gingras group reports 129 proteins in FLAG alone negative controls on a more sensitive mass spectrometer, the TripleTOF 5600, and this list only includes peptides detected by two or more peptides (see Supplemental Table 2B in ref 40). We have an average of 147 proteins detected by two or more peptides in our three FLAG alone negative controls in Supplemental Table 6 (Supporting Information) using a more sensitive mass spectrometer, the LTQ, which is consistent with the work done by the Gingras group.40 As a result, our FLAG alone control data agrees with previously published studies.

Table 1. Detection of RNA Polymerase Subunits by HaloTag and FLAG Affinity Systemsa complex

protein

HT mock

S (I−III) S (I−III) S (I−III) S (I−III) S (I−III) I and III I and III I I I I I I II II II II II II III III III III III III III III III III III

POLR2E POLR2F POLR2H POLR2K POLR2L RPAC1 RPAC2 POLR1A POLR1B POLR1E hRPA34 RPA43 RPA12 POLR2A POLR2B POLR2C POLR2D POLR2I POLR2J POLR3A POLR3B POLR3C POLR3D POLR3E POLR3F POLR3G POLR3GL POLR3H POLR3I POLR3K

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

HT POLR2H 3 2 3 1 3 3 3 3 3 1 3 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 2 1 2 3

of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

FLAG mock n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1of 3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

FLAG POLR2H 1 of n.d. 3 of n.d. n.d. 3 of n.d. 2 of 1 of n.d. 2 of n.d. n.d. 3 of 3 of 2 of 1 of n.d. n.d. 1 of n.d. 1 of 2 of n.d. n.d. n.d. n.d. n.d. n.d. n.d.

3 3

3 3 3 3

Identification of Interacting Partners and Discovery of Novel Interacting Partners

3 3 3 3

In addition to the core subunits, several other known associated factors important for RNAP activity were also identified via POL2RH (Figure 4), suggesting that in vivo, the POL2RH fusions are incorporated into active complexes. These proteins include RuvB1, RuvB2, RPAP2, and RPAP3 and have been shown to interact with RNAP II.29 The capture of interacting partners within the HT-POLR2H expression deletion series followed the trend that decreased expression lead to decreased capture efficiency of non-core RNAP proteins (Figure 4, Supporting Table 2, Supporting Information). Interestingly, three novel RNAP interacting proteins, MLLT1141 and two proteins of unknown function LOC85395 and LOC90488, were consistently identified in the high, medium, and low HTPOLR2H pulldowns and not in the HaloTag alone control (Figure 4, Supporting Table 2, Supporting Information). To confirm these interactions were specific and also identify with which polymerase they were associated, each was fused to HaloTag and reciprocal pulldowns were performed and analyzed using quantitative proteomics (Figure 5). These data show the proteins most abundant in the pull-downs for each of these three novel proteins are known interacting partners of RNAP II, including RuvB-like1 and 2 helicase proteins or Elongin B and C (Figure 5). Only a few core subunits were identified from RNAP II and even fewer from RNAP I, suggesting these proteins likely are secondary or tertiary interactors of RNA polymerase core components (Figure 5 and Supporting Table 4, Supporting Information). Strikingly, these three proteins isolate each other as well as very similar interacting partners (Figure 5), suggestive that they are either

3 3 3

a

In Complex column, S (I−III) indicates the subunit is shared between RNA Polymerases I, II, and III. “I and III” indicates that the subunit is shared between RNA Polymerase I and III. I, II, and III correspond to components of RNA Polymerase I, II, and III, respectively. Also, n.d. is not detected. X of 3 indicates number of replicates detected and identified in. All data is from the same number of starting cells (1.2 × 107). HT-POLR2H data is from pFN21A cell lines (High expression), which has the same intact CMV promoter as the FLAG-POLR2H cell line. The data presented in this table is a summary of data from Supporting Table 2 (HT-POLR2H) and Supporting Table 8 (FLAG POLR2H), Supporting Information.

purification of FLAG-POLR2H HEK293T cells detected and identified 4 RNAP subunits in 3 of 3 replicates, 4 RNAP subunits in 2 of 3 replicates, and 5 RNAP subunits in 1 of 3 570



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DISCUSSION

We show here the ability to isolate and identify three macromolecular complexes, the eukaryotic RNA polymerases I, II, and III, and associated proteins in a single pull-down using the HaloTag technology coupled with quantitative proteomics. Expression studies demonstrate the incorporation of the HTPOLR2H fusion into expected RNA polymerase complexes across a wide variety of expression levels with similar levels of background among the samples. In addition to the protein complex isolations, correlative cellular localization of the HaloTag fusions using fluorescent HaloTag ligands demonstrate proper localization to the nucleus as well as discovery of localization patterns of three previously unknown RNA polymerase associated proteins identified and validated in the HaloTag pull-down assays. The ability to efficiently isolate protein complexes utilizing the HaloTag technology as demonstrated by these studies is likely due to the unique features of the HaloTag fusion protein.11 The first feature, the rapid binding of the HaloTag fusion protein to its ligands, was obtained and optimized using directed evolution to have a final on-rate similar to that of biotin-streptavidin.11 This short binding time allows for HaloTag fusion proteins, even those involved in large complexes such as the RNA polymerases, to be captured on its resin within a time frame of 15−30 min as compared to the typical time of 1−2 h needed when performing an immunoprecipitation. The ability to rapidly capture and concentrate complexes onto a surface kinetically favors complex integrity, and promotes maintenance of weak and/or transient interactions. The second unique feature is the irreversible binding of HaloTag to its ligand, which further favors complex integrity, as complexes are covalently held and concentrated on the resin, without being lost to diffusion. The covalent capture may also be beneficial for capture of low abundance complexes and may be the primary reason why even with near endogenous level expression, isolation of the RNA polymerases was very efficient. Together, these two properties of HaloTag, not common to antibodies or other affinity tags, maximize the chance to keep complexes in tact after cellular lysis and throughout the purification and allows for the use of a minimal number of cells for the purification process. Equally important to the maintenance of complexes during the isolation is the minimization of background or nonspecific interactions. As the coupled affinity tag/mass spectrometry analysis expands in technical and quantitative capacities, reduction of the number of contaminants or nonspecific proteins in samples is critical for identification of specific interactions. High levels of peptides from contaminating proteins can mask peptides of true interacting partners in the mass spectrometry analysis. Analysis of the HaloTag pull-down samples revealed the overall background contaminants within the samples to be low and RNAP core subunits to be consistently detected with similar dNSAF values. The significant advantage of coupling HaloTag pull-downs with proteomic approaches is the ability to elute interacting partners without eluting the HaloTag fusion protein. When using any overexpressed protein, there will be some cellular population which is uncomplexed and potentially even more efficiently captured on a resin than those in complexes due to steric hindrance. If eluted, this uncomplexed population can become problematic, acting as a contaminant and obscuring the identification of other specific proteins.

Figure 4. Quantitative mass spectrometry analysis of associated RNAP proteins from HT-POLR2H complex isolations. POLR2H subcloned into pFN21A CMV (high expression, black bars), pFN22K CMVd1 (medium expression, red bars), pFN23K CMVd2 (low expression, blue bars), and pFN24K CMVd3 (ultra low expression, pink bars) HaloTag Flexi Vectors and expressed in HEK293T cells and were affinity purified and analyzed using quantitative proteomics approaches. Shown are dNSAF values of POL2RH associated proteins and all values are avg ± std deviation of three biological replicates.

directly associated with each or part of a shared complex. Interestingly, LOC85395 differs from the three in that one of the most prominent proteins unique in its pull-downs is human Transportin-1 (Figure 5 and Supporting Table 4), a 100 kDa βimportin family member protein important for nuclear import of several hnRNP proteins and transcription factors.42 Cellular Localization Studies

Demonstrating the multifunctionality of the HaloTag, the same constructs that were used for affinity purification analysis were used to perform cellular imaging studies with the HaloTag fusions of POLR2H, MLLT11, LOC85395, and LOC90488 by addition of the HaloTag TMR fluorescent ligand to the media of cells expressing the respective constructs (Figure 6). To show colocalization of the HaloTag fusion proteins with endogenous RNAP II, standard immunocytochemistry staining experiments were performed using an antibody against the POLR2A C-terminal domain (CTD) (Figure 6). The HTPOLR2H showed primary localization to the nucleus, matching that of POLR2A28 and correlating well with the pull-down data (Figure 6A). The three novel interactors however revealed significantly different localization patterns than POLR2H. MLLT11 and LOC90488 both showed both localization to the cytoplasm and nucleus, with matching nuclear localization patterns as POLR2A (Figure 6B,D). Whether or not these proteins have a cytoplasmic function remains to be discovered, but the pull-down data did show several cytoplasmic proteins as possible interactors (Supporting Table 4, Supporting Information). Interestingly again, LOC85395 is different from the other two proteins as it was restricted to the nucleus, though unlike POLR2A, showed localization throughout the nucleus to include the nucleolar staining (Figure 6C). Whether or not this is due to its unique and strong interaction with Transportin-1 remains to be determined. 571



Article

Figure 5. Reciprocal complex isolations and mass spectrometry analysis of identified interacting proteins. MLLT11 (black bars), LOC85395 (red bars), and LOC90488 (blue bars) were subcloned into pFN21A CMV (high expression) HaloTag Flexi Vector and expressed in HEK293 cells and were affinity purified and analyzed using quantitative proteomics approaches. (A) Silver stained SDS gel of proteins eluted after the HaloTag complex isolation protocol for MLLT11 (Lane 1), LOC85395 (Lane 2), LOC90488 (Lane 3), and HaloTag alone control (Lane 4). (B) Shown are dNSAF values of bait proteins. (C) Shown are dNSAF values of RNA Polymerase II subunits. (D) Shown are dNSAF values of associated proteins. All values are avg ± stnd deviation of two biological replicates.

pulldown to investigate any aberrant changes to physiology. These data across the expression series showed the HTPOLR2H fusion protein incorporated into expected RNA polymerase complexes and was properly localized to the nucleus. It is also important to note the RNA polymerase could be efficiently captured at expression levels similar to endogenous, which might be necessary for some proteins in maintaining physiological relevant complexes. The expression series pull-downs coupled with the quantitative mass spectrometry also allowed for the relative abundance of each polymerase and its subunits to be determined. Interestingly, subunits from RNAP II were in the highest concentration, followed by RNAP III and I respectively across the HaloTag expression series, (Figure 2). While it is possible that the POLR2H fusion proteins incorporates more efficiently into

The use of any exogenously expressed protein fusion has the ability to disrupt biological processes due to positioning or size of the tag, which could sterically hinder proper complex formation, or also levels of overexpression inside the cell. HaloTag, 34kD, is similar in size to GFP which has been used extensively for cellular localization studies and most recently for protein complex isolations.9 As either the size or placement of HaloTag could potentially be problematic, it was important to simultaneously confirm proper complex formation and localization. It would not be surprising if N- or C-terminal placement of the HaloTag for similar types of studies could be crucial to obtaining physiological complexes, and there may also be examples where neither termini can be successfully tagged. To address the concerns of protein overexpression, the CMV promoter deletion series was performed for the HaloTag 572



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Figure 6. Cellular localization of HaloTag fusion proteins and colocalization with endogenous POLR2A. Confocal cellular images of U20S cells expressing (A) HT-POLR2H, (B) HT-MLLT11, (C) HT-LOC85395, and (D) HT-LOC90488. The top left panel in A−D shows localization of endogenous POLR2A protein detected using an anti-POLR2A antibody in conjunction with an Alexa 488 fluorescent secondary antibody. The top right panel in A−D shows localization of the respective HaloTag fusion protein labeled covalently with a red TMR fluorescent HaloTag ligand. The bottom right panel in A−D is the overlay of the top panels to demonstrate colocalization. The bottom left panel in A−D is the corresponding bright field image of the cells.

activation. Further research as to which genes are either up- or down-regulated would be important for furthering understanding of how high levels of MLLT11 might be connected to AML. As this protein is also found to be localized to the cytoplasm, it may have other cytoplasmic interactions important for its function. Homology searches with LOC85395 and LOC90488 do not reveal any clues as to what their function may be. They show very distinct localization patterns, and similar to MLLT11, LOC90488 may have some important role or interaction in the cytoplasm as it is found there as well. LOC85395, which has a strong interaction also with Transportin-1, may play dual roles in the nucleus, regulation of RNAP II and also regulation of import via the nuclear pore complex.42 Further experiments remain to better understand the function of both these unknown proteins.

RNAP II as compared to the others, this may reflect the true relative abundance of each RNA polymerase in the cell. The RNA polymerases interact with hundreds of different protein partners directly or indirectly, though many of these complexes are labile as RNA polymerases need to rapidly switch between different activity states. As mentioned above, RNAP II was the most abundant polymerase isolated, therefore it was not surprising the associated proteins discovered were associated toward RNAP II. While several known and expected associated proteins were identified, the discovery of MLLT11 and two unknown proteins, LOC85395 and LOC90488 demonstrate the sensitivity of the HaloTag technology for discovery of new interactions. By reciprocal tagging these three proteins with HaloTag, each were validated back to an interaction with RNAP, though likely indirectly with associated proteins of RNAP II and may potentially interact with each other. Of the three proteins, only MLLT11 has a known function and was initially identified as a mixed-lineage leukemia (MLL) fusion partner arising from a t(1;11) (q21;23) translocation.41,43 This protein is highly expressed in patients with acute myeloid leukemia (AML), yet the functional consequence of this increased expression remains unknown.41,43 The link to RNAP II is intriguing and suggests a potential role of this protein in transcriptional regulation and

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CONCLUSIONS This comprehensive and quantitative proteomics study of isolation of the eukaryotic RNA polymerases from mammalian cells combined with cellular imaging highlights the utility of the versatile HaloTag technology for proteomics research and protein interaction network analyses. The RNA polymerases were chosen for these studies not only to highlight the strengths of the technology, but also with goals to further 573



Article

(6) Ewing, R. M.; Chu, P.; Elisma, F.; Li, H.; Taylor, P.; Climie, S.; McBroom-Cerajewski, L.; Robinson, M. D.; O’Connor, L.; Li, M.; Taylor, R.; Dharsee, M.; Ho, Y.; Heilbut, A.; Moore, L.; Zhang, S.; Ornatsky, O.; Bukhman, Y. V.; Ethier, M.; Sheng, Y.; Vasilescu, J.; Abu-Farha, M.; Lambert, J. P.; Duewel, H. S.; Stewart, I. I.; Kuehl, B.; Hogue, K.; Colwill, K.; Gladwish, K.; Muskat, B.; Kinach, R.; Adams, S. L.; Moran, M. F.; Morin, G. B.; Topaloglou, T.; Figeys, D. Large-scale mapping of human protein-protein interactions by mass spectrometry. Mol. Syst. Biol. 2007, 3, 89. (7) Behrends, C.; Sowa, M. E.; Gygi, S. P.; Harper, J. W. Network organization of the human autophagy system. Nature 2010, 466 (7302), 68−76. (8) Sowa, M. E.; Bennett, E. J.; Gygi, S. P.; Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 2009, 138 (2), 389−403. (9) Hutchins, J. R.; Toyoda, Y.; Hegemann, B.; Poser, I.; Heriche, J. K.; Sykora, M. M.; Augsburg, M.; Hudecz, O.; Buschhorn, B. A.; Bulkescher, J.; Conrad, C.; Comartin, D.; Schleiffer, A.; Sarov, M.; Pozniakovsky, A.; Slabicki, M. M.; Schloissnig, S.; Steinmacher, I.; Leuschner, M.; Ssykor, A.; Lawo, S.; Pelletier, L.; Stark, H.; Nasmyth, K.; Ellenberg, J.; Durbin, R.; Buchholz, F.; Mechtler, K.; Hyman, A. A.; Peters, J. M. Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science 2010, 328 (5978), 593−9. (10) Gavin, A. C.; Maeda, K.; Kuhner, S. Recent advances in charting protein-protein interaction: mass spectrometry-based approaches. Curr. Opin. Biotechnol. 2011, 22 (1), 42−9. (11) Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 2008, 3 (6), 373−82. (12) von Mering, C.; Krause, R.; Snel, B.; Cornell, M.; Oliver, S. G.; Fields, S.; Bork, P. Comparative assessment of large-scale data sets of protein-protein interactions. Nature 2002, 417 (6887), 399−403. (13) Nguyen, T. N.; Goodrich, J. A. Protein-protein interaction assays: eliminating false positive interactions. Nat. Methods 2006, 3 (2), 135−9. (14) Ohana, R. F.; Hurst, R.; Vidugiriene, J.; Slater, M. R.; Wood, K. V.; Urh, M. HaloTag-based purification of functional human kinases from mammalian cells. Protein Expr. Purif. 2011, 76 (2), 154−64. (15) Hartzell, D. D.; Trinklein, N. D.; Mendez, J.; Murphy, N.; Aldred, S. F.; Wood, K.; Urh, M. A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays. BMC Genomics 2009, 10, 497. (16) Woychik, N. A.; Liao, S. M.; Kolodziej, P. A.; Young, R. A. Subunits shared by eukaryotic nuclear RNA polymerases. Genes Dev. 1990, 4 (3), 313−23. (17) Mosley, A. L.; Sardiu, M. E.; Pattenden, S. G.; Workman, J. L.; Florens, L.; Washburn, M. P. Highly reproducible label free quantitative proteomic analysis of RNA polymerase complexes. Mol. Cell. Proteomics 2011, 10 (2), M110 000687. (18) Sims, R. J., 3rd; Mandal, S. S.; Reinberg, D. Recent highlights of RNA-polymerase-II-mediated transcription. Curr. Opin. Cell Biol. 2004, 16 (3), 263−71. (19) Lee, T. I.; Young, R. A. Transcription of eukaryotic proteincoding genes. Annu. Rev. Genet. 2000, 34, 77−137. (20) Narlikar, G. J.; Fan, H. Y.; Kingston, R. E. Cooperation between complexes that regulate chromatin structure and transcription. Cell 2002, 108 (4), 475−87. (21) Sims, R. J., 3rd; Belotserkovskaya, R.; Reinberg, D. Elongation by RNA polymerase II: the short and long of it. Genes Dev. 2004, 18 (20), 2437−68. (22) Coulombe, B.; Jeronimo, C.; Langelier, M. F.; Cojocaru, M.; Bergeron, D. Interaction networks of the molecular machines that decode, replicate, and maintain the integrity of the human genome. Mol. Cell. Proteomics 2004, 3 (9), 851−6. (23) White, R. J. RNA polymerases I and III, growth control and cancer. Nat. Rev. Mol. Cell. Biol. 2005, 6 (1), 69−78.

understanding of RNAP research through discovery of new protein interactions. The identification of three novel interaction proteins and their cellular localization patterns demonstrated this was possible and will lead to future research experiments. Here we demonstrated the ability of the HaloTag to efficiently capture protein complexes and the use the same constructs for cellular localization studies. Therefore, the HaloTag approach may serve to accelerate the analysis of protein complex and protein interaction network analyses.

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Danette L. Daniels Promega Corporation 2800 Woods Hollow Road Madison, WI 53711. Phone: 608-274-4330. Fax: 608-277-2601. E-mail: [email protected]. Michael P. Washburn Stowers Institute for Medical Research 1000 E. 50th St. Kansas City, MO 64110. Phone: 816-9264392. Fax: 816-926-4692. E-mail: [email protected].

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ACKNOWLEDGMENTS This work was supported by the Stowers Institute for Medical Research and Promega Corporation. Promega Corporation sells the HaloTag mammalian pull-down interaction system and fluorescent HaloTag imaging ligands commercially. Some of the Promega authors hold stock in Promega Corporation, but less than 1% of such stock. Promega Corporation is the owner by assignment of patents or patent applications related to the HaloTag technology.

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REFERENCES

(1) Tyers, M.; Mann, M. From genomics to proteomics. Nature 2003, 422 (6928), 193−7. (2) Cravatt, B. F.; Simon, G. M.; Yates, J. R. 3rd The biological impact of mass-spectrometry-based proteomics. Nature 2007, 450 (7172), 991−1000. (3) Gavin, A. C.; Aloy, P.; Grandi, P.; Krause, R.; Boesche, M.; Marzioch, M.; Rau, C.; Jensen, L. J.; Bastuck, S.; Dumpelfeld, B.; Edelmann, A.; Heurtier, M. A.; Hoffman, V.; Hoefert, C.; Klein, K.; Hudak, M.; Michon, A. M.; Schelder, M.; Schirle, M.; Remor, M.; Rudi, T.; Hooper, S.; Bauer, A.; Bouwmeester, T.; Casari, G.; Drewes, G.; Neubauer, G.; Rick, J. M.; Kuster, B.; Bork, P.; Russell, R. B.; Superti-Furga, G. Proteome survey reveals modularity of the yeast cell machinery. Nature 2006, 440 (7084), 631−6. (4) Krogan, N. J.; Cagney, G.; Yu, H.; Zhong, G.; Guo, X.; Ignatchenko, A.; Li, J.; Pu, S.; Datta, N.; Tikuisis, A. P.; Punna, T.; Peregrin-Alvarez, J. M.; Shales, M.; Zhang, X.; Davey, M.; Robinson, M. D.; Paccanaro, A.; Bray, J. E.; Sheung, A.; Beattie, B.; Richards, D. P.; Canadien, V.; Lalev, A.; Mena, F.; Wong, P.; Starostine, A.; Canete, M. M.; Vlasblom, J.; Wu, S.; Orsi, C.; Collins, S. R.; Chandran, S.; Haw, R.; Rilstone, J. J.; Gandi, K.; Thompson, N. J.; Musso, G.; St Onge, P.; Ghanny, S.; Lam, M. H.; Butland, G.; Altaf-Ul, A. M.; Kanaya, S.; Shilatifard, A.; O’Shea, E.; Weissman, J. S.; Ingles, C. J.; Hughes, T. R.; Parkinson, J.; Gerstein, M.; Wodak, S. J.; Emili, A.; Greenblatt, J. F. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 2006, 440 (7084), 637−43. (5) Sardiu, M. E.; Cai, Y.; Jin, J.; Swanson, S. K.; Conaway, R. C.; Conaway, J. W.; Florens, L.; Washburn, M. P. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (5), 1454−9. 574



Article

(24) Thompson, N. E.; Aronson, D. B.; Burgess, R. R. Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyolresponsive monoclonal antibody. J. Biol. Chem. 1990, 265 (12), 7069− 77. (25) Cloutier, P.; Al-Khoury, R.; Lavallee-Adam, M.; Faubert, D.; Jiang, H.; Poitras, C.; Bouchard, A.; Forget, D.; Blanchette, M.; Coulombe, B. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes. Methods 2009, 48 (4), 381−6. (26) Domecq, C.; Kireeva, M.; Archambault, J.; Kashlev, M.; Coulombe, B.; Burton, Z. F. Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II. Protein Expr. Purif. 2010, 69 (1), 83−90. (27) Jeronimo, C.; Langelier, M. F.; Zeghouf, M.; Cojocaru, M.; Bergeron, D.; Baali, D.; Forget, D.; Mnaimneh, S.; Davierwala, A. P.; Pootoolal, J.; Chandy, M.; Canadien, V.; Beattie, B. K.; Richards, D. P.; Workman, J. L.; Hughes, T. R.; Greenblatt, J.; Coulombe, B. RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits. Mol. Cell. Biol. 2004, 24 (16), 7043−58. (28) Sugaya, K.; Vigneron, M.; Cook, P. R. Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein. J. Cell Sci. 2000, 113 (Pt 15), 2679−83. (29) Forget, D.; Lacombe, A. A.; Cloutier, P.; Al-Khoury, R.; Bouchard, A.; Lavallee-Adam, M.; Faubert, D.; Jeronimo, C.; Blanchette, M.; Coulombe, B. The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II. Mol. Cell. Proteomics 2010, 9 (12), 2827−39. (30) Jeronimo, C.; Forget, D.; Bouchard, A.; Li, Q.; Chua, G.; Poitras, C.; Therien, C.; Bergeron, D.; Bourassa, S.; Greenblatt, J.; Chabot, B.; Poirier, G. G.; Hughes, T. R.; Blanchette, M.; Price, D. H.; Coulombe, B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol. Cell 2007, 27 (2), 262−74. (31) Cramer, P.; Bushnell, D. A.; Fu, J.; Gnatt, A. L.; Maier-Davis, B.; Thompson, N. E.; Burgess, R. R.; Edwards, A. M.; David, P. R.; Kornberg, R. D. Architecture of RNA polymerase II and implications for the transcription mechanism. Science 2000, 288 (5466), 640−9. (32) Elias, J. E.; Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 2007, 4 (3), 207−14. (33) Tabb, D. L.; McDonald, W. H.; Yates, J. R. 3rd DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 2002, 1 (1), 21−6. (34) Zhang, Y.; Wen, Z.; Washburn, M. P.; Florens, L. Refinements to label free proteome quantitation: how to deal with peptides shared by multiple proteins. Anal. Chem. 2010, 82 (6), 2272−81. (35) Werner, M.; Thuriaux, P.; Soutourina, J. Structure-function analysis of RNA polymerases I and III. Curr. Opin. Struct. Biol. 2009, 19 (6), 740−5. (36) Panov, K. I.; Panova, T. B.; Gadal, O.; Nishiyama, K.; Saito, T.; Russell, J.; Zomerdijk, J. C. RNA polymerase I-specific subunit CAST/ hPAF49 has a role in the activation of transcription by upstream binding factor. Mol. Cell. Biol. 2006, 26 (14), 5436−48. (37) Haurie, V.; Durrieu-Gaillard, S.; Dumay-Odelot, H.; Da Silva, D.; Rey, C.; Prochazkova, M.; Roeder, R. G.; Besser, D.; Teichmann, M. Two isoforms of human RNA polymerase III with specific functions in cell growth and transformation. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (9), 4176−81. (38) Einhauer, A.; Jungbauer, A. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J. Biochem. Biophys. Methods 2001, 49 (1−3), 455−65. (39) Einhauer, A.; Jungbauer, A. Affinity of the monoclonal antibody M1 directed against the FLAG peptide. J. Chromatogr., A 2001, 921 (1), 25−30.

(40) Dunham, W. H.; Larsen, B.; Tate, S.; Badillo, B. G.; Goudreault, M.; Tehami, Y.; Kislinger, T.; Gingras, A. C. A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples. Proteomics 2011, 11 (13), 2603−12. (41) Tse, W.; Meshinchi, S.; Alonzo, T. A.; Stirewalt, D. L.; Gerbing, R. B.; Woods, W. G.; Appelbaum, F. R.; Radich, J. P. Elevated expression of the AF1q gene, an MLL fusion partner, is an independent adverse prognostic factor in pediatric acute myeloid leukemia. Blood 2004, 104 (10), 3058−63. (42) Pollard, V. W.; Michael, W. M.; Nakielny, S.; Siomi, M. C.; Wang, F.; Dreyfuss, G. A novel receptor-mediated nuclear protein import pathway. Cell 1996, 86 (6), 985−94. (43) Busson-Le Coniat, M.; Salomon-Nguyen, F.; Hillion, J.; Bernard, O. A.; Berger, R. MLL-AF1q fusion resulting from t(1;11) in acute leukemia. Leukemia 1999, 13 (2), 302−6.

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Examining the Complexity of Human RNA Polymerase Complexes

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