The Myotonic Dystrophy Type 2 Protein ZNF9 Is Part of an ITAF Complex That Promotes Cap-independent Translation
2007; Elsevier BV; Volume: 6; Issue: 6 Linguagem: Inglês
10.1074/mcp.m600384-mcp200
ISSN1535-9484
AutoresVincent R. Gerbasi, Andrew J. Link,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe 5′-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy. The 5′-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy. Most eukaryotic translation initiation involves the interaction of the 43 S preinitiation complex (comprised of the 40 S ribosomal subunit plus initiation factors, Met-tRNAi, and GTP) with the 7-methylguanosine cap complex at the 5′-end of the mRNA (1Sachs A.B. Sarnow P. Hentze M.W. Starting at the beginning, middle, and end: translation initiation in eukaryotes.Cell. 1997; 89: 831-838Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). During such cap-dependent translation initiation, the 43 S preinitiation complex is recruited to the 5′-cap structure and scans the 5′-untranslated region (UTR). 1The abbreviations used are: UTR, untranslated region; IRES, internal ribosomal entry site; ITAF, IRES trans-acting factor; ODC, ornithine decarboxylase; BIGCAT, Bioinformatic Graphical Comparative Analysis Tools; PAF, protein abundance factor; CAT, chloramphenicol acetyltransferase; siRNA, small interfering RNA; EMSA, electrophoretic mobility shift assay; RNP, ribonucleoprotein; IP, immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PY, pyrimidine; RE, regulatory element; PTBP, polypyrimidine tract-binding protein; UNR, upstream of N-ras. Recognition of the AUG start codon is followed by joining of the 40 S-Met-tRNAi complex to the 60 S large ribosomal subunit to form the 80 S initiation complex. In contrast, translation mediated by an internal ribosomal entry site (IRES) does not require the 5′-cap structure. Instead translation initiates at internal sites in the mRNA (2Pelletier J. Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.Nature. 1988; 334: 320-325Crossref PubMed Scopus (1395) Google Scholar, 3Jang S.K. Davies M.V. Kaufman R.J. Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5′ nontranslated region of encephalomyocarditis virus RNA in vivo.J. Virol. 1989; 63: 1651-1660Crossref PubMed Google Scholar, 4Chen C.Y. Sarnow P. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs.Science. 1995; 268: 415-417Crossref PubMed Scopus (506) Google Scholar). IRESs are cis-acting RNA sequences found in the 5′-region of a subset of eukaryotic mRNAs. Originally discovered in viral mRNAs, IRESs were later found in cellular transcripts (5Yang Q. Sarnow P. Location of the internal ribosome entry site in the 5′ non-coding region of the immunoglobulin heavy-chain binding protein (BiP) mRNA: evidence for specific RNA-protein interactions.Nucleic Acids Res. 1997; 25: 2800-2807Crossref PubMed Scopus (86) Google Scholar). It is postulated that 3–5% of all human mRNAs are translated in a cap-independent manner (6Johannes G. Carter M.S. Eisen M.B. Brown P.O. Sarnow P. Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13118-13123Crossref PubMed Scopus (323) Google Scholar, 7Qin X. Sarnow P. Preferential translation of internal ribosome entry site-containing mRNAs during the mitotic cycle in mammalian cells.J. Biol. Chem. 2004; 279: 13721-13728Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Some translational initiation factors used in cap-dependent initiation also stimulate translation of some, but not all, IRES-containing transcripts. There are examples of IRES-containing mRNAs that appear to recruit ribosomes directly, independently of the classical initiation factors (8Ji H. Fraser C.S. Yu Y. Leary J. Doudna J.A. Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16990-16995Crossref PubMed Scopus (145) Google Scholar, 9Wilson J.E. Pestova T.V. Hellen C.U. Sarnow P. Initiation of protein synthesis from the A site of the ribosome.Cell. 2000; 102: 511-520Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 10Hellen C.U. Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules.Genes Dev. 2001; 15: 1593-1612Crossref PubMed Scopus (805) Google Scholar). Each IRES may use a unique mechanism of translational initiation. Evidence from studies of both viral and cellular IRESs has led to several hypotheses as to how and why IRESs initiate translation of their cognate mRNAs. First, IRESs may be an efficient alternative to cap-dependent translation initiation, an observation supported by multiple studies (6Johannes G. Carter M.S. Eisen M.B. Brown P.O. Sarnow P. Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13118-13123Crossref PubMed Scopus (323) Google Scholar, 7Qin X. Sarnow P. Preferential translation of internal ribosome entry site-containing mRNAs during the mitotic cycle in mammalian cells.J. Biol. Chem. 2004; 279: 13721-13728Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 11Johannes G. Sarnow P. Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.RNA. 1998; 4: 1500-1513Crossref PubMed Scopus (222) Google Scholar, 12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Second, specific RNA-binding proteins may be dedicated to facilitating IRES-mediated translation initiation. This model has gained favor through the discovery that viral and cellular IRESs share a common set of RNA-binding proteins that stimulate their translation (13Walter B.L. Nguyen J.H. Ehrenfeld E. Semler B.L. Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements.RNA. 1999; 5: 1570-1585Crossref PubMed Scopus (121) Google Scholar, 14Blyn L.B. Swiderek K.M. Richards O. Stahl D.C. Semler B.L. Ehrenfeld E. Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5′ noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11115-11120Crossref PubMed Scopus (171) Google Scholar, 15Blyn L.B. Towner J.S. Semler B.L. Ehrenfeld E. Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA.J. Virol. 1997; 71: 6243-6246Crossref PubMed Google Scholar, 16Evans J.R. Mitchell S.A. Spriggs K.A. Ostrowski J. Bomsztyk K. Ostarek D. Willis A.E. Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.Oncogene. 2003; 22: 8012-8020Crossref PubMed Scopus (186) Google Scholar, 17Hunt S.L. Hsuan J.J. Totty N. Jackson R.J. unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA.Genes Dev. 1999; 13: 437-448Crossref PubMed Scopus (220) Google Scholar, 18Mitchell S.A. Brown E.C. Coldwell M.J. Jackson R.J. Willis A.E. Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras.Mol. Cell. Biol. 2001; 21: 3364-3374Crossref PubMed Scopus (130) Google Scholar). Finally there is evidence that a subset of IRESs can recruit ribosomes directly through a complex RNA secondary structure (9Wilson J.E. Pestova T.V. Hellen C.U. Sarnow P. Initiation of protein synthesis from the A site of the ribosome.Cell. 2000; 102: 511-520Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 19Pestova T.V. Hellen C.U. Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA.Genes Dev. 2003; 17: 181-186Crossref PubMed Scopus (185) Google Scholar). The sequences and structures of viral and cellular IRESs vary widely although they often contain pyrimidine-rich sequences. The conserved pyrimidine tract is found proximal to the start codon (1Sachs A.B. Sarnow P. Hentze M.W. Starting at the beginning, middle, and end: translation initiation in eukaryotes.Cell. 1997; 89: 831-838Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). Mutagenesis of pyrimidines proximal to the start codon or in other locations throughout an IRES reduces activity and results in the disruption of specific protein-RNA interactions (12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 20Mitchell S.A. Spriggs K.A. Bushell M. Evans J.R. Stoneley M. Le Quesne J.P. Spriggs R.V. Willis A.E. Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry.Genes Dev. 2005; 19: 1556-1571Crossref PubMed Scopus (104) Google Scholar). Thus, these pyrimidine-rich sequences in IRESs are necessary for complete IRES activity and recruitment of trans-acting factors. The IRES trans-acting factor PTBP binds pyrimidine-rich sequences in viral and cellular IRESs (18Mitchell S.A. Brown E.C. Coldwell M.J. Jackson R.J. Willis A.E. Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras.Mol. Cell. Biol. 2001; 21: 3364-3374Crossref PubMed Scopus (130) Google Scholar, 20Mitchell S.A. Spriggs K.A. Bushell M. Evans J.R. Stoneley M. Le Quesne J.P. Spriggs R.V. Willis A.E. Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry.Genes Dev. 2005; 19: 1556-1571Crossref PubMed Scopus (104) Google Scholar, 21Singh R. Valcarcel J. Green M.R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.Science. 1995; 268: 1173-1176Crossref PubMed Scopus (465) Google Scholar). It functions with UNR, another ITAF, to enhance the activity of viral and cellular IRESs (17Hunt S.L. Hsuan J.J. Totty N. Jackson R.J. unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA.Genes Dev. 1999; 13: 437-448Crossref PubMed Scopus (220) Google Scholar, 18Mitchell S.A. Brown E.C. Coldwell M.J. Jackson R.J. Willis A.E. Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras.Mol. Cell. Biol. 2001; 21: 3364-3374Crossref PubMed Scopus (130) Google Scholar). In addition, several other RNA-binding proteins have been shown to bind and enhance the activity of IRESs (22Stoneley 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 (291) Google Scholar). However, the high sequence variability between IRESs suggests that there is a wide array of ITAFs, many of which are unknown. Pancreatic tumor cells alternatively splice the 5′-UTR of ODC to generate an IRES that is translated in a cap-independent and cell cycle-dependent manner (12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 23Pyronnet S. Pradayrol L. Sonenberg N. Alternative splicing facilitates internal ribosome entry on the ornithine decarboxylase mRNA.Cell. Mol. Life Sci. 2005; 62: 1267-1274Crossref PubMed Scopus (18) Google Scholar, 24Pyronnet S. Vagner S. Bouisson M. Prats A.C. Vaysse N. Pradayrol L. Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5′ untranslated region.Cancer Res. 1996; 56: 1742-1745PubMed Google Scholar). Pyrimidine tracts in the ODC 5′-UTR that are necessary for IRES activity have been identified by site-directed mutagenesis (12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Mutations that disrupt either RNA secondary structure or interactions with trans-acting factors compromise IRES activity. As such, we chose the ODC IRES as the target in our search for novel proteins that modulate cellular IRES activity. To identify potential proteins that associate with the ODC IRES, we utilized a proteomics approach (25Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2074) Google Scholar). Using RNA affinity capture and mass spectrometry, we found that two nucleic acid-binding proteins, PCBP2 and ZNF9, associate with the wild-type ODC IRES. Mutations in the IRES sequence that compromise ODC IRES function reduced binding to these two proteins. PCBP2 is a known IRES-binding protein that enhances cap-independent translation (13Walter B.L. Nguyen J.H. Ehrenfeld E. Semler B.L. Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements.RNA. 1999; 5: 1570-1585Crossref PubMed Scopus (121) Google Scholar, 14Blyn L.B. Swiderek K.M. Richards O. Stahl D.C. Semler B.L. Ehrenfeld E. Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5′ noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11115-11120Crossref PubMed Scopus (171) Google Scholar, 15Blyn L.B. Towner J.S. Semler B.L. Ehrenfeld E. Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA.J. Virol. 1997; 71: 6243-6246Crossref PubMed Google Scholar, 16Evans J.R. Mitchell S.A. Spriggs K.A. Ostrowski J. Bomsztyk K. Ostarek D. Willis A.E. Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.Oncogene. 2003; 22: 8012-8020Crossref PubMed Scopus (186) Google Scholar, 26Gamarnik A.V. Andino R. Interactions of viral protein 3CD and poly(rC) binding protein with the 5′ untranslated region of the poliovirus genome.J. Virol. 2000; 74: 2219-2226Crossref PubMed Scopus (185) Google Scholar). The function of ZNF9 is unknown, although its non-coding region is mutated in patients with type 2 myotonic dystrophy (27Liquori C.L. Ricker K. Moseley M.L. Jacobsen J.F. Kress W. Naylor S.L. Day J.W. Ranum L.P. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.Science. 2001; 293: 864-867Crossref PubMed Scopus (1009) Google Scholar). Our results suggest that one function of ZNF9 is to enhance cap-independent translation. 3′-Biotinylated RNAs used for affinity capture reactions were purchased from Dharmacon Inc. The wild-type ODC RNA sequence was 5′-UUUCUGUCUUAUUGUUUC-3′ (12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The mutant ODC RNA sequence was 5′-AAACUGUCUUAUUGAAAC-3′ (12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). RAJI human B-cell lymphocytes were grown in RPMI 1640 medium, 10% FCS, 1% penicillin-streptomycin at 37 °C and 5% CO2. Human 293T cells were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 1% penicillin-streptomycin at 37 °C and 5% CO2. Antibodies raised against PCBP2 were kindly provided by Raul Andino. α-V5 antibodies were purchased from Invitrogen. α-Protein kinase C and α-actin antibodies were purchased from Santa Cruz Biotechnology Inc. To clone the ZNF9 cDNA, total RNA was isolated from human RAJI B-cells. cDNA was generated using Superscript II (Invitrogen) primed with oligo(dT). The ZNF9 cDNA was amplified using the primers 5′-GGCAAGGACCCTCAAAATAAAC-3′ (forward) and 5′-TGTAGCCTCAATTGTGCATTC-3′ (reverse). The 620-bp RT-PCR product was cloned in-frame into the pcDNA3.1/V5-His-TOPO plasmid to create the plasmid pcDNA-ZNF9-V5 expressing a ZNF9-V5 fusion. The plasmid pcDNA3.1/V5-His-TOPO/lacZ expressing a lacZ-V5 fusion was obtained from Invitrogen. To generate the RNA affinity chromatography resin, 100 μl of streptavidin-agarose beads (Pierce) were incubated with 30 nmol of wild-type or mutant 3′-biotinylated RNA in binding buffer (12 mm HEPES in diethyl pyrocarbonate-treated water, pH 8.0, 15 mm KCl, 15 mm dithiothreitol, 5 mm MgCl2, 10% glycerol with one mini-Complete™ protease inhibitor tablet (Roche Applied Science)/50 ml of binding buffer) at 4 °C for 30 min with gentle mixing. Following incubation of biotinylated RNA with the streptavidin beads, the chromatography resin was washed with binding buffer (200× bead volumes) to remove excess biotinylated RNA. To generate cell extracts, 109 cells were suspended in 3 ml of binding buffer and lysed in 2-ml tubes with 0.5-mm glass beads using a bead beater (Biospec, Inc). The supernatant was removed from the glass beads into sterile microcentrifuge tubes and centrifuged at 20,000 × g for 15 min. The cleared supernatant was incubated with the agarose-coupled RNA affinity resin for 30 min at 4 °C with gentle mixing. After incubation, the chromatography resin was washed with binding buffer (200× bead volumes) to remove nonspecific proteins. Proteins were eluted from the RNA affinity resin with 1-ml washes of increasing salt concentration (150, 250, 350, and 600 mm NaCl) in binding buffer. Each salt wash was then subjected to a 10% TCA precipitation. Protein pellets were suspended in 100 mm ammonium bicarbonate prior to SDS-PAGE, trypsinization, and mass spectrometry analysis. Experiments utilizing a competitive, non-biotinylated RNA were performed essentially as described above with the exception that a wild-type RNA affinity column was incubated with cell extract and then washed twice with 30 nmol of non-biotinylated ODC RNA suspended in 1 ml of binding buffer. Proteins that remained associated with the resin were eluted with four separate salt washes (150, 250, 350, and 600 mm NaCl) and prepared for mass spectrometry analysis as described below. Eluted proteins from the RNA affinity capture experiments were reduced, alkylated, and trypsinized in 100 mm ammonium bicarbonate as described previously (28Sanders S.L. Jennings J. Canutescu A. Link A.J. Weil P.A. Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry.Mol. Cell. Biol. 2002; 22: 4723-4738Crossref PubMed Scopus (262) Google Scholar). The tryptic peptide fragments were desalted using a C18 reverse-phase salt trap (Michrom) and were subjected to reverse-phase microcapillary LC-ESI-MS/MS. A fritless, microcapillary column (100-μm inner diameter) was packed with 10 cm of 5-μm C18 reverse-phase material (Synergi 4u Hydro RP80a, Phenomenex). The trypsin-digested peptides were loaded onto the reverse-phase column equilibrated in buffer A (0.1% formic acid, 5% acetonitrile). The column was placed in line with an LTQ linear ion trap mass spectrometer (Thermo Electron, Inc.). Peptides were eluted using a 60-min linear gradient from 0 to 60% buffer B (0.1% formic acid, 80% acetonitrile) at a flow rate of 0.3 μl/min. During the gradient, the eluted ions were analyzed by one full precursor MS scan (400–2000 m/z) followed by five MS/MS scans of the five most abundant ions detected in the precursor MS scan while operating under dynamic exclusion. The program extractms2 was used to generate the ASCII peak list and identify +1 or multiply charged precursor ions from the native mass spectrometry data file. 2J. Eng and J. R. Yates III, unpublished software. Tandem spectra were searched with no protease specificity using SEQUEST-PVM (29Sadygov R.G. Eng J. Durr E. Saraf A. McDonald H. MacCoss M.J. Yates III, J.R. Code developments to improve the efficiency of automated MS/MS spectra interpretation.J. Proteome Res. 2002; 1: 211-215Crossref PubMed Scopus (183) Google Scholar) against the RefSeq human protein database (released May 2005) containing 28,818 entries. For multiply charged precursor ions (z ≥ +2), an independent search was performed on both the +2 and +3 mass of the parent ion. Data were processed and organized using the BIGCAT software analysis suite (30McAfee K.J. Duncan D.T. Assink M. Link A.J. Analyzing proteomes and protein function using graphical comparative analysis of tandem mass spectrometry results.Mol. Cell. Proteomics. 2006; 5: 1497-1513Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). A weighted scoring matrix was used to select the most likely charge state of multiply charged precursor ions (25Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2074) Google Scholar, 30McAfee K.J. Duncan D.T. Assink M. Link A.J. Analyzing proteomes and protein function using graphical comparative analysis of tandem mass spectrometry results.Mol. Cell. Proteomics. 2006; 5: 1497-1513Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). From the database search, fully tryptic peptide sequences with Sequest cross-correlation scores ≥1.5 for +1 ions, ≥2 for +2 ions, and ≥2 for +3 ions were considered significant and used to create the list of identified proteins. For the proteins ZNF9 and PCBP2 pursued in this study, the annotated MS/MS spectra identifying the two proteins are available in Supplemental Fig. 3. A complete listing of all identified peptides and proteins along with the relevant scoring metrics is available in Supplemental Tables 1 and 2. To estimate the relative abundance of a protein from the mass spectrometry data, a protein abundance factor (PAF) was calculated for each identified protein (31Powell D.W. Weaver C.M. Jennings J.L. McAfee K.J. He Y. Weil P.A. Link A.J. Cluster analysis of mass spectrometry data reveals a novel component of SAGA.Mol. Cell. Biol. 2004; 24: 7249-7259Crossref PubMed Scopus (124) Google Scholar, 32Fleischer T.C. Weaver C.M. McAfee K.J. Jennings J.L. Link A.J. Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes.Genes Dev. 2006; 20: 1294-1307Crossref PubMed Scopus (219) Google Scholar). To calculate PAF values, the total number of non-redundant spectra that correlated significantly with each cognate protein was normalized to the molecular weight of the protein (×104) (31Powell D.W. Weaver C.M. Jennings J.L. McAfee K.J. He Y. Weil P.A. Link A.J. Cluster analysis of mass spectrometry data reveals a novel component of SAGA.Mol. Cell. Biol. 2004; 24: 7249-7259Crossref PubMed Scopus (124) Google Scholar, 32Fleischer T.C. Weaver C.M. McAfee K.J. Jennings J.L. Link A.J. Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes.Genes Dev. 2006; 20: 1294-1307Crossref PubMed Scopus (219) Google Scholar). Protein bands corresponding to the predicted molecular weights of PCBP2 and ZNF9 were excised from silver-stained gels and sliced into 1-mm cubes. The gel pieces were dehydrated in acetonitrile and then rehydrated in 100 mm ammonium bicarbonate. After rehydration, the samples were brought to a 1:1 equal volume of ammonium bicarbonate and acetonitrile. Gel pieces were lyophilized to dryness. The samples were suspended in digestion buffer (50 mm ammonium bicarbonate, 0.5 mm CaCl2, and 0.0125 μg/μl trypsin). The gel pieces remained in digestion buffer for 45 min on ice. Following incubation, 20 μl of additional digestion buffer without trypsin was added, and the gel pieces were incubated for 18 h at 37 °C. Trypsinized peptides were extracted three times from the gel pieces with 50-μl washes of 25 mm ammonium bicarbonate and acetonitrile (1:1). The pooled supernatants were frozen and lyophilized. The dried peptides were resuspended in 10 μl of 0.1% formic acid and subjected to the LC-ESI-MS/MS analysis described above. Sucrose gradient analysis was performed as described previously (33Link A.J. Fleischer T.C. Weaver C.M. Gerbasi V.R. Jennings J.L. Purifying protein complexes for mass spectrometry: applications to protein translation.Methods. 2005; 35: 274-290Crossref PubMed Scopus (27) Google Scholar). To analyze the activity of the bicistronic reporter (12Pyronnet S. Pradayrol L. Sonenberg N. A cell cycle-dependent internal ribosome entry site.Mol. Cell. 2000; 5: 607-616Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), 293T cells were transfected with either 10 μg of the bicistronic reporter plus 10 μg of pcDNA-ZNF9-V5 or 10 μg of the bicistronic reporter plus 10 μg of pcDNA3.1/V5-His-TOPO/lacZ. Each transfection mixture contained 60 μl of Lipofectamine 2000 (Invitrogen). All reporter assays were performed in triplicate. All transfections were performed in Opti-MEM minimal medium as recommended by the manufacturer (Invitrogen). After 4 h of transfection, the Opti-MEM medium was replaced with full medium containing serum. Cells were harvested 48 h after transfection and assayed for luciferase and chloramphenicol acetyltransferase (CAT) activity (34Nordeen S.K. Green III, P.P. Fowlkes D.M. A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase.DNA. 1987; 6: 173-178Crossref PubMed Scopus (178) Google Scholar). Reporter assays using RNA interference were similar to the assays described above. On day 1, 293T cells were simultaneously transfected with 10 μg of the bicistronic reporter and 400 nm siRNAs specific for either lamin A/C (35Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8161) Google Scholar), ZNF9 (5′-GCUAUUCUUGUGGAGAAUU-3′), PCBP2 (5′-GCAUUCCACAAUCCAUCAUUU-3′), or a combination of ZNF9 and PCBP2 siRNAs using 60 μl of LipofectAMINE 2000. All siRNAs used in this study were purchased from Dharmacon Inc. siRNAs for PCBP2 targeted both isoforms detected in our mass spectrometry analysis. On day 2, 293T cells were retransfected with a 400 nm concentration of the same siRNAs except that 60 μl of Oligofectamine/transfection was used instead of the LipofectAMINE 2000. Twenty-four hours after the second transfection, the cells were harvested and assayed for luciferase and CAT activity. Experiments were performed in triplicate. A Student's two-tailed t-test was performed to test the statistical significance of the difference between the experimental and control results. Electrophoretic mobility shift assays (EMSAs) were performed essentially as described previously (36Rychlik J.L. Gerbasi V. Lewis E.J. The interaction between dHAND and Arix at the dopamine beta-hydroxylase promoter region is independent of direct dHAND binding to DNA.J. Biol. Chem. 2003; 278: 49652-49660Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We used oligoribonucleotides corresponding to the wild-type ODC IRES RNA sequence (5′-UUUCUGUCUUAUUGUUUC-3′) and the mu
Referência(s)