Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach
2011; Elsevier BV; Volume: 10; Issue: 4 Linguagem: Inglês
10.1074/mcp.m110.007385
ISSN1535-9484
AutoresBecky Pinjou Tsai, Xiaorong Wang, Lan Huang, Marian L. Waterman,
Tópico(s)RNA modifications and cancer
ResumoIdentification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes. Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes. RNA-protein complexes play central roles in post-transcriptional regulation, but their dynamic nature can make it challenging to identify protein components and define steps in RNA processes. This is especially true for internal ribosome entry site (IRES) 1The abbreviations used are:IRESinternal ribosome entry siteHB-tagHistidine and biotin tagMS2-HBbacteriophage MS2 coat protein dimer fused to a HTBH-tagLEF1Lymphoid Enhancer Factor-1MS2-BioTRAPMS2 in vivo Biotin Tagged RNA Affinity PurificationSILACStable isotope labeling with amino acids in cell cultureITAFIRES trans-acting factorsCapIndicates canonical cap-dependent translationMAPmix after purificationPAM-SILACpurification after mixing. elements. IRESs are long stretches of noncoding RNA in the 5′ untranslated (UTR) regions of a subset of cellular and viral mRNAs (1Fitzgerald K.D. Semler B.L. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.Biochim. Biophys. Acta. 2009; 1789: 518-528Crossref PubMed Scopus (136) Google Scholar, 2Jackson R.J. Hellen C.U. Pestova T.V. The mechanism of eukaryotic translation initiation and principles of its regulation.Nat. Rev. Mol. Cell Biol. 2010; 11: 113-127Crossref PubMed Scopus (1772) Google Scholar, 3Pacheco A. Martinez-Salas E. Insights into the biology of IRES elements through riboproteomic approaches.J. Biomed. Biotechnol. 2010; 2010458927Crossref PubMed Scopus (53) Google Scholar, 4Stoneley M. Willis A.E. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (292) Google Scholar). Unlike most mRNAs which have short 5′UTRs, the highly structured regions of the LEF1 IRES and other lengthy IRESs impede ribosome scanning (1Fitzgerald K.D. Semler B.L. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.Biochim. Biophys. Acta. 2009; 1789: 518-528Crossref PubMed Scopus (136) Google Scholar, 3Pacheco A. Martinez-Salas E. Insights into the biology of IRES elements through riboproteomic approaches.J. Biomed. Biotechnol. 2010; 2010458927Crossref PubMed Scopus (53) Google Scholar, 4Stoneley M. Willis A.E. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (292) Google Scholar). Ribosome recruitment and translation of mRNAs with short 5′UTRs rely on the canonical cap-binding complex (eIF4F) which recognizes a 7-methylguanosine cap structure on the 5′ of eukaryotic mRNAs (1Fitzgerald K.D. Semler B.L. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.Biochim. Biophys. Acta. 2009; 1789: 518-528Crossref PubMed Scopus (136) Google Scholar, 2Jackson R.J. Hellen C.U. Pestova T.V. The mechanism of eukaryotic translation initiation and principles of its regulation.Nat. Rev. Mol. Cell Biol. 2010; 11: 113-127Crossref PubMed Scopus (1772) Google Scholar, 4Stoneley M. Willis A.E. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (292) Google Scholar). In the case of viral mRNAs, which often do not have a 7-methylguanosine cap, IRESs can capture ribosomes for translation via IRES trans-acting factors (ITAFs) or specific RNA secondary structures (5Balvay L. Soto Rifo R. Ricci E.P. Decimo D. Ohlmann T. Structural and functional diversity of viral IRESs.Biochim. Biophys. Acta. 2009; 1789: 542-557Crossref PubMed Scopus (143) Google Scholar). In the case of IRESs in eukaryotic, cellular mRNAs, the mechanism for recruitment of ribosomes can function either as an alternative to canonical cap-dependent recruitment mechanisms when translation is compromised (e.g. nutrient deprivation, hypoxia, and mitosis), or as a cap-enhancing mechanism to increase translation (1Fitzgerald K.D. Semler B.L. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.Biochim. Biophys. Acta. 2009; 1789: 518-528Crossref PubMed Scopus (136) Google Scholar, 3Pacheco A. Martinez-Salas E. Insights into the biology of IRES elements through riboproteomic approaches.J. Biomed. Biotechnol. 2010; 2010458927Crossref PubMed Scopus (53) Google Scholar, 6Gilbert W.V. Alternative ways to think about cellular internal ribosome entry.J. Biol. Chem. 2010; 285: 29033-29038Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). internal ribosome entry site Histidine and biotin tag bacteriophage MS2 coat protein dimer fused to a HTBH-tag Lymphoid Enhancer Factor-1 MS2 in vivo Biotin Tagged RNA Affinity Purification Stable isotope labeling with amino acids in cell culture IRES trans-acting factors Indicates canonical cap-dependent translation mix after purification purification after mixing. IRESs were originally discovered in RNA viruses such as picornaviruses. Viral IRESs are several hundred nucleotides in length, their sequence is highly conserved, and they form tightly folded RNA scaffolds for ITAF assembly and ribosome interactions (5Balvay L. Soto Rifo R. Ricci E.P. Decimo D. Ohlmann T. Structural and functional diversity of viral IRESs.Biochim. Biophys. Acta. 2009; 1789: 542-557Crossref PubMed Scopus (143) Google Scholar). Many of these RNA structures and interacting ITAFs are well studied. Unlike viral IRESs, our understanding of cellular IRESs is limited. Cellular IRESs are estimated to be present in 3–5% of capped mRNA transcripts, they are highly variable in length (up to several kilobases) and they do not exhibit the same degree of sequence conservation compared with viral IRESs (4Stoneley M. Willis A.E. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (292) Google Scholar). These features make the identification of structures and components of cellular IRES-protein complexes extremely challenging. Conventional strategies of IRES-protein analysis involve in vitro approaches with synthetic RNA, aptamer tags, and in vitro purification. These approaches have identified a handful of IRES trans-acting factors (ITAFs), which are overwhelmingly represented by hnRNPs and other abundant RNA binding proteins (1Fitzgerald K.D. Semler B.L. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.Biochim. Biophys. Acta. 2009; 1789: 518-528Crossref PubMed Scopus (136) Google Scholar, 4Stoneley M. Willis A.E. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (292) Google Scholar, 7Cobbold L.C. Spriggs K.A. Haines S.J. Dobbyn H.C. Hayes C. de Moor C.H. Lilley K.S. Bushell M. Willis A.E. Identification of internal ribosome entry segment (IRES)-trans-acting factors for the Myc family of IRESs.Mol. Cell. Biol. 2008; 28: 40-49Crossref PubMed Scopus (110) Google Scholar). IRES-protein complexes are dynamic structures and their compositions are subject to change depending on many factors including RNA folding, subcellular localization, and transcription/post-transcription processes that modify the RNP/mRNA complex as it moves from the nucleus through nuclear pores to sites of translation in the cytoplasm (1Fitzgerald K.D. Semler B.L. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.Biochim. Biophys. Acta. 2009; 1789: 518-528Crossref PubMed Scopus (136) Google Scholar, 8Fujimura K. Kano F. Murata M. Identification of PCBP2, a facilitator of IRES-mediated translation, as a novel constituent of stress granules and processing bodies.Rna. 2008; 14: 425-431Crossref PubMed Scopus (62) Google Scholar, 9Lewis S.M. Veyrier A. Hosszu Ungureanu N. Bonnal S. Vagner S. Holcik M. Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation.Mol. Biol. Cell. 2007; 18: 1302-1311Crossref PubMed Scopus (94) Google Scholar). Thus, although they are useful, in vitro purification strategies are not best suited for the capture of in vivo assembled IRES-protein complexes. In order to preserve authentic RNA-protein complexes as they are isolated from living cells, several new methods have recently been developed (10Nonne N. Ameyar-Zazoua M. Souidi M. Harel-Bellan A. Tandem affinity purification of miRNA target mRNAs (TAP-Tar).Nucleic Acids Res. 2010; 38: e20Crossref PubMed Scopus (55) Google Scholar, 11Hafner M. Landthaler M. Burger L. Khorshid M. Hausser J. Berninger P. Rothballer A. Ascano Jr., M. Jungkamp A.C. Munschauer M. Ulrich A. Wardle G.S. Dewell S. Zavolan M. Tuschl T. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.Cell. 2010; 141: 129-141Abstract Full Text Full Text PDF PubMed Scopus (2134) Google Scholar, 12Tyagi S. Imaging intracellular RNA distribution and dynamics in living cells.Nat. Methods. 2009; 6: 331-338Crossref PubMed Scopus (327) Google Scholar, 13Butter F. Scheibe M. Mörl M. Mann M. Unbiased RNA-protein interaction screen by quantitative proteomics.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 10626-10631Crossref PubMed Scopus (106) Google Scholar, 14Hogg J.R. Collins K. RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation.Rna. 2007; 13: 868-880Crossref PubMed Scopus (119) Google Scholar). These strategies are protein-centric in that a specific RNA binding protein is tagged, expressed in vivo, and used as bait to capture its interacting RNAs for subsequent microarray analysis or deep sequencing. These strategies require a known RNA binding protein and do not allow effective identification of other components in RNA-protein complexes at a proteome scale. To circumvent this problem, we have developed an integrated proteomic strategy that is RNA-centric and uses MS2 in vivo Biotin Tagged RNA Affinity Purification (MS2-BioTRAP) and stable isotope labeling with amino acid in cell culture (SILAC)-based quantitative mass spectrometry. In this strategy, a specific RNA is tagged with a cluster of RNA stem-loops recognized by bacteriophage protein MS2, an RNA binding protein that binds to the single-stranded loop region with nanomolar affinity (15Keryer-Bibens C. Barreau C. Osborne H.B. Tethering of proteins to RNAs by bacteriophage proteins.Biol. Cell. 2008; 100: 125-138Crossref PubMed Scopus (67) Google Scholar, 16Lim F. Spingola M. Peabody D.S. Altering the RNA binding specificity of a translational repressor.J. Biol. Chem. 1994; 269: 9006-9010Abstract Full Text PDF PubMed Google Scholar, 17Hook B. Bernstein D. Zhang B. Wickens M. RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening.Rna. 2005; 11: 227-233Crossref PubMed Scopus (93) Google Scholar). MS2 is HB-tagged and co-expressed for in vivo association with the stem-loop tagged RNA (18Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.Mol. Cell Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 19Wang X. Chen C.F. Baker P.R. Chen P.L. Kaiser P. Huang L. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.Biochemistry. 2007; 46: 3553-3565Crossref PubMed Scopus (206) Google Scholar). The HB tag consists of two hexahistidine tags, a TEV cleavage site, and a signal sequence for in vivo biotinylation (19Wang X. Chen C.F. Baker P.R. Chen P.L. Kaiser P. Huang L. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.Biochemistry. 2007; 46: 3553-3565Crossref PubMed Scopus (206) Google Scholar). This enables rapid and effective one-step purification of MS2-HB, its associated stem-loop tagged RNA, and all other proteins bound to the tagged RNA. To maintain the integrity of protein-RNA complexes during the purification processes, in vivo UV cross-linking is carried out prior to cell lysis to freeze RNA-protein interactions in living cells. SILAC-based quantitative mass spectrometry is subsequently employed to quantitatively identify proteins associating with specific IRES RNAs in comparison with a non-IRES RNA (e.g. Cap) control sample. The results have been further validated by co-immunoprecipitation, quantitative Western blot, and siRNA knock-down experiments to demonstrate that MS2-BioTRAP captures bona fide interactors that regulate the LEF1 IRES. The work presented here describes a general proteomic strategy that is valuable for studying in vivo RNA-protein complexes as they occur in living cells. Dicistronic reporter plasmids pRstF and pRstF-5′UTR (20Jimenez J. Jang G.M. Semler B.L. Waterman M.L. An internal ribosome entry site mediates translation of lymphoid enhancer factor-1.Rna. 2005; 11: 1385-1399Crossref PubMed Scopus (33) Google Scholar) were used to generate tagged-Cap and tagged-IRES expression constructs, respectively. To generate a monocistronic reporter plasmid, the NheI and EcoRI sites were used to remove the upstream Renilla luciferase open reading frame and bisect and destroy the subsequent stem-loop. The circular plasmid was regenerated by blunt end ligation. The monocistronic reporters were then linearized (XbaI site) between the Firefly luciferase stop codon and poly(A) signal sequence and a MS2 stem-loop fragment containing four tandem stem-loops was inserted by blunt end ligation (MS2 stem-loop template, SP73-βglobin-(MS2)4, was a gift from Klemens Hertel). The MS2 coat protein sequence was amplified from pCT119-N55K (gift from David Peabody, University of New Mexico) using a three-piece ligation strategy. To generate a tandem-linked dimer of open reading frames, the first MS2 coat protein in the dimer was generated by PCR amplification of the MS2 coat protein plasmid sequence using a sense primer (5′-AATCTGAGCGGCCGCGCATGGCTTCTAACTTTACTCA-3′) containing a NotI site (italicized) upstream of MS2 sequence and an antisense primer containing a BglI site downstream of the MS2 sequence (5′-ATTCAGCCGTAGAGGCCGGAGTTTGCTGCGATT-3′). The second MS2 coat protein was amplified using a sense primer (5′-ACTCAGGCCTCTACGGCGCAATGGCTTCTAACTTTACTCA-3′) containing a BglI site upstream of the MS2 sequence end and an antisense primer (5′-CCTTAATTAAGGAGTTTGCTGCGATT-3′) containing a PacI site downstream of the MS2 sequence. BglI restriction enzyme sites at the 3′ and 5′ end of each PCR product allowed for blunt end ligation and creation of a tail-to-head tandem placement of two MS2 coat protein open reading frames. The two PCR products were digested with the indicated restriction enzymes and cloned into the HTBH (a derivative of HB tag) tag vector (pQCXIP backbone (18Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.Mol. Cell Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 19Wang X. Chen C.F. Baker P.R. Chen P.L. Kaiser P. Huang L. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.Biochemistry. 2007; 46: 3553-3565Crossref PubMed Scopus (206) Google Scholar)) linearized with NotI and PacI. Two hundred and ninety-three stable cell lines expressing MS2-HB (293MS2-HB) were generated by retrovirus infection as described (19Wang X. Chen C.F. Baker P.R. Chen P.L. Kaiser P. Huang L. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.Biochemistry. 2007; 46: 3553-3565Crossref PubMed Scopus (206) Google Scholar). Briefly, 293GP2 packaging cells were plated at a density of 7 × 106 cells/10 cm-diameter tissue culture dish and transfected with pQCXIP-MS2-HB retroviral vector using a calcium phosphate protocol. Following 8 h, the medium was replaced with fresh Dulbecco's modified Eagle's medium (DMEM). Thirty-six hours post-transfection, the conditioned medium containing the retroviruses was collected every 8 h for 60 h. Two hundred and ninety-three cells were infected by incubation with equal volumes of fresh DMEM and retrovirus conditioned medium and 4 μg/ml of polybrene. Following 4–6 h, cells were washed and a second infection was performed. Thirty hours postinfection, cells were seeded at 2 × 106 cells/10-cm plate and cultured in DMEM containing the selection antibiotic puromycin (3 μg/ml). Following ∼5 days of selection, cells were seeded at a density of 2 × 102 to 2 × 103 cells per 10 cm-diameter tissue culture dish. Individual clones were picked from the plates and expanded to generate stable cell clones expressing MS2-HB. The stable cell line expressing MS2-HB, 293MS2-HB, was grown in SILAC DMEM (Thermo Scientific, #89985) supplemented with 28 μg/ml 12C614N4-arginine, 73 μg/ml 12C614N2-lysine (Sigma) (light medium) or 13C615N4-arginine and 13C615N2-lysine (heavy medium) purchased from Cambridge Isotope Laboratories (Andover, MA), 10% fetal bovine serum, 1% penicillin/streptomycin, 3 μg/ml puromycin (stable cell selection), and 5 μm biotin (Sigma). Cell lines were grown for more than seven cell doublings in the labeling media to ensure complete incorporation. The cells were then grown to confluence prior to cell lysis. Two hundred and ninety-threeMS2-HB cells were cultured in 150-mm plates and transfected with the respective tagged-RNAs as described (21Wang X. Huang L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry.Mol. Cell Proteomics. 2008; 7: 46-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 22Oeffinger M. Wei K.E. Rogers R. DeGrasse J.A. Chait B.T. Aitchison J.D. Rout M.P. Comprehensive analysis of diverse ribonucleoprotein complexes.Nat. Methods. 2007; 4: 951-956Crossref PubMed Scopus (205) Google Scholar, 23Ule J. Jensen K. Mele A. Darnell R.B. CLIP: a method for identifying protein-RNA interaction sites in living cells.Methods. 2005; 37: 376-386Crossref PubMed Scopus (450) Google Scholar). Forty-eight hours post-transfection, cells were washed with ice-cold phosphate-buffered saline (PBS), cultures were then immersed in 7 ml 1× PBS, and UV cross-linked using 2400 Stratalinker to irradiate one time for 400 mJ/cm2. Cells were harvested by scraping and collected by centrifugation. Cell pellets were lysed using either the native lysis buffer L (100 mm NaCl, 50 mm Tris, 5 mm MgCl2, 10% glycerol, 0.5% NP-50, RNasIN (Optizyme Ribonuclease Inhibitor, Fisher, 5000×), 1 mm PMSF, Protease Inhibitor (Sigma, 1000×), 50 mm NaF, 0.1 mm Na4VO4, 5 mm EDTA, 5 mm EGTA, 0.5 mm β-mercaptoethanol)) or denaturing lysis buffer A-8 (8 m Urea, 300 mm NaCl, 50 mm NaH2PO4/NaHPO4, 0.5% Nonidet P-40) (lysis volumes were chosen to achieve 15–20 mg/ml total protein). Lysate was sonicated using the Branson Sonifier 450 (Setting Duty 50%, Output 4) three times for 15-s intervals. Lysate was centrifuged to remove large debris and clarified by filtration through 1.6 μm GD/X Glass Microfiber filters (Whatman). All steps were performed on ice, with ice-cold reagents. The purification procedure is similar to a previously reported procedure (19Wang X. Chen C.F. Baker P.R. Chen P.L. Kaiser P. Huang L. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.Biochemistry. 2007; 46: 3553-3565Crossref PubMed Scopus (206) Google Scholar, 22Oeffinger M. Wei K.E. Rogers R. DeGrasse J.A. Chait B.T. Aitchison J.D. Rout M.P. Comprehensive analysis of diverse ribonucleoprotein complexes.Nat. Methods. 2007; 4: 951-956Crossref PubMed Scopus (205) Google Scholar). Briefly, Dynal streptavidin M-280 magnetic beads (Invitrogen) were prepared by washing three times with the same buffer used for cell lysis. Amount of beads used varied with amount of total protein in the lysate (∼1 μl bead slurry/20 μg lysate). For native purification, we employed the MAP-SILAC method (mix after purification) to isolate RNA-protein complexes from light and heavy labeled lysates separately to prevent interaction exchange during purification (21Wang X. Huang L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry.Mol. Cell Proteomics. 2008; 7: 46-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Each lysate was applied to beads in a 1.5-ml tube and rotated at 4 °C for 5 min. An ice-cold magnet was applied to the side of the tube to isolate the beads along with the captured RNP complexes. The flow-through lysate was removed and the beads were washed on ice 2× with ∼50× bed volume of the same buffer used for cell lysis. For on-bead trypsin digestion, beads were washed again in ice-cold 25 mm NH4CO3 (∼30× bed volume) and resuspended with 5–10 ng/μl Promega (Madison, WI) trypsin (diluted in 25 mm NH4CO3). Digestion of the sample proceeded at 37 °C for 8–12 h. For denaturing conditions, equal amounts of lysate were mixed from the light and heavy conditions, followed by addition of the magnetic beads (PAM-SILAC, purification after mixing) (21Wang X. Huang L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry.Mol. Cell Proteomics. 2008; 7: 46-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The steps thereafter were the same as in native purification. Optional TEV digestion and elution: For native purification, TEV (Tobacco Etch Virus Protease; Invitrogen) digestion can be used to release MS2-HB and captured RNA/protein complexes. Following the wash step with lysis buffer, complexes were washed again with 30× bed volume of ice-cold TEB buffer (50 mM Tris-HCl pH 8.0, 10% glycerol, 0.5 mM EDTA, 1% Triton X-100, 1 mm DTT), then incubated with two bed volumes of TEV (0.25 U/μl, diluted with TEB) for 1 h at room temperature. A magnet was applied to collect the elution and followed with trypsin digestion. Tryptic digests were first separated by strong cation exchange chromatography using a polysulfoethyl A column (2.1 mm i.d. × 10 cm long) (Nest Group) at a flow rate of 200 μl/min using AKTA Basic 10 (GE Healthcare) (21Wang X. Huang L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry.Mol. Cell Proteomics. 2008; 7: 46-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Peptide elution was achieved by a salt gradient (0–350 mm KCl), and fractions were manually collected based on UV absorbance at 215 nm. The collected fractions were desalted using Vivapure C18 microspin columns (Vivascience, Aubagne, France) prior to LC MS/MS. LC-MS/MS was carried out by nanoflow reversed-phase liquid chromatography (RPLC) (Eksigent, Dublin, CA) coupled online to a linear ion trap (LTQ)-Orbitrap XL mass spectrometer (Thermo-Electron Corp) (24Kaake R.M. Milenković T. Przulj N. Kaiser P. Huang L. Characterization of cell cycle specific protein interaction networks of the yeast 26S proteasome complex by the QTAX strategy.J. Proteome Res. 2010; 9: 2016-2029Crossref PubMed Scopus (49) Google Scholar). The LC analysis was performed using a capillary column (100 μm i.d. × 150 mm long) packed with Inertsil ODS-3 resin (GL. Sciences, CA); the peptides were eluted using a linear gradient of 2% to 35% B in 85 min at a flow rate of 350 μl/min (solvent A, 100% H2O-0.1% formic acid; solvent B, 100% acetonitrile-0.1% formic acid). A cycle of one full Fourier transform scan mass spectrum (350–1800 m/z, resolution of 60,000 at m/z 400) followed by 10 data-dependent MS/MS acquired in the linear ion trap with normalized collision energy (setting of 35%). Target ions already selected for MS/MS were dynamically excluded for 30 s. The MS data was extracted and analyzed as described (24Kaake R.M. Milenković T. Przulj N. Kaiser P. Huang L. Characterization of cell cycle specific protein interaction networks of the yeast 26S proteasome complex by the QTAX strategy.J. Proteome Res. 2010; 9: 2016-2029Crossref PubMed Scopus (49) Google Scholar). Monoisotopic masses of parent ions and corresponding fragment ions, parent ion charge states, and ion intensities from LC-MS/MS spectra were extracted using in-house software based on Raw_Extract script from Xcalibur v2.4. Following automated data extraction, the resultant peak lists for each LC-MS/MS experiment were submitted to the development version (5.3.0) of Protein Prospector (UCSF) for database searching using a concatenated Swissprot database (857302 sequence entries) composed of a SwissProt database (3/24/2009) and its randomized version.. Homo sapiens was selected as the restricted species. Trypsin was set as the enzyme with a maximum of two missed cleavage sites. The mass tolerances for parent and fragment ions were set as 20 ppm and 0.8 Da respectively. Chemical modifications such as protein N-terminal acetylation, methionine oxidation, N-terminal pyroglutamine, and deamidation of asparagine were selected as variable modifications. For SILAC experiments, 13C615N4-Arg and 13C615N2-Lys were also chosen as variable modifications. The Search Compare program in Protein Prospector was used for summarization, validation, and comparison of results. A false positive (% FP) rate of ≤0.2% was used for peptide identification calculated in Search Compare (24Kaake R.M. Milenković T. Przulj N. Kaiser P. Huang L. Characterization of cell cycle specific protein interaction networks of the yeast 26S proteasome complex by the QTAX strategy.J. Proteome Res. 2010; 9: 2016-2029Crossref PubMed Scopus (49) Google Scholar). At this fals
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