Proteomic Analysis of Bovine Nucleolus

Available online at www.sciencedirect.comGENOMICSPROTEOMICS &ScienceDirectBIOINFORMATICSEL SEVIERwww.sciencedirect.com/science/journal/16720229ArticleProteomic Analysis of Bovine NucleolusAmrutlal K. Patel',, Doug Olson', and Suresh K. Tikol,2.4*' Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada;'Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada;National Research Council, Plant Biotechnology Institute, University of Saskatchewan, Saskatoon, Canada;4School of Public Health, University of Saskatchewan, Saskatoon, Canada.Genomics Proteomics Bioinformatics 2010 Sep; 8(3): 145-158 DOI: 10.1016/S1672-0229(10)60017-4AbstractNucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomic composition of the bovine nucleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subected to proteomic analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nucleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.Key words: bovine, nucleolus, proteomics, 1D SDS- PAGE, SCX, LC-MS/MSIntroductionribosome biogenesis, nucleolus plays a wide range ofcellular functions, which include biogenesis of ribo-Nucleolus is a dynamic subnuclear domain, whichnucleoproteins (13, 14) and signal recognition parti-cles (I5, 16), regulation of the cell cycle (17-20),forms around the clusters of ribosomal DNA duringRNA editing (I7, 21-23)， stress response (24, 25),the interphase of the cell cycle (1-6). The mammaliancell contains 1-6 nucleoli with varying sizes andregulation of telomerase activity (26), tRNA process-numbers, depending upon the cell types and cultureing (27, 28), aging (29), apoptosis (30), export of nu-clear proteins (3I) and mRNAs (32), and viral repli-conditions, even within the same cell (I, 7). The nu-cation (33-36).cleolus was traditionally known to be the ribo-The nucleoli are formed as a result of the ribosomalsome-producing apparatus resulting from the act ofbuilding the ribosomes (8). However, research overgene transcription, processing of 47S pre-ribosomalthe past decade explored its functional complexityRNA (rRNA) into 5.8S, 18S, and 28S rRNAs, andbeyond manufacturing of ribosomes (9-12). Besidesribosomal subunit assembly (I, 3, 8). The nucleoliconsist of three distinct sub-compartments, namely*Corresponding author.fibrillar centres (FCs), dense fibrillar centres (DFCs)E-mail: suresh.tik@usask.caand granular c中国煤化工37, 38). TheC 2010 Beijing Institute of Genomics. All rights reserved.rRNAs are syrYHC N M H GaccumulatedPatel et al. /Proteomic Analysis of Bovine Nucleolusand processed in the DFCs, and assemble with theResultsribosomal proteins and 5S rRNAs to form the ribo-somal subunits in the GCs. The nucleolar assemblyIsolation of nucleoli from bovine cellsstarts at the end of mitosis with the reinitiation of theribosomal gene transcription and recruitment of theThe nucleoli were isolated from Madin Darby bovinerRNA processing components from the prenucleolarkidney (MDBK) cells and analyzed for their puritybodies in the nucleolar organizer regions (NORs;and integrity by various methods. For nucleoli isola-cluster of rDNA repeats) on different chromosomestion from MDBK cells, the addition of 0.3% NP-40 in(39, 40). These NORs associate to form one or morethe hypotonic lysis buffer improved the disruption offunctional nucleoli. At the onset of the prophase, thethe cell membrane and separation of the cytoplasmicRNA polymerase I transcription and pre-rRNA proc-material from the nucleus. The nucleolar integrity wasessing are repressed. The DFCs and the GCs startanalyzed under light microscope after each sonicationsegregating and disappearing at the end of prophase,step to obtain the efficient nuclear disruption withleading to the disassembly of the nucleolus (40, 41).minimum loss of the nucleoli. The purity of the nu-The nucleolar proteins continuously shuttle betweencleolar preparation was analyzed by Western blot.the nucleoli and the nucleoplasm (42-44). However,Proteins from equal amount of cytoplasmic, nucleo-the proteins having the specific interactions with theplasmic and nucleolar fractions were separated onnucleolar components are retained in the nucleoplasm10% SDS- PAGE (sodium dodecyl sulfate poly-for longer duration (45). The dynamic flux of the nu-acrylamide gel electrophoresis), transferred to thecleolar proteins has also been revealed by the proteo-nitrocellulose membrane and probed with anti-ERK2mic analysis of the nucleoli under various metabolicserum, anti-Nup62 serum and anti-nucleolin/fibrillarinconditions (46). Although not enclosed by membrane,serum recognizing a cytoplasm, a nucleoplasm and adue to its unique density and the ability to retain itsnucleolar specific protein, respectively. As seen instructural integrity following sonication, the nucleoliFigure 1A, anti-ERK2 serum recognized a specificcan be isolated by the sucrose density gradient cen-protein in the cytoplasmic and nucleoplasmic frac-trifugation (2, 47-50).tions but not in the nucleolar fraction. Similarly,Recently, the proteomic analysis of human (46, 47,anti-Nup62 serum recognized a specific protein in the49) and plant nucleoli (51) not only has helped to gainnucleoplasmic and cytoplasmic fractions but not ininsights into the nucleolar organization and function, .the nucleolar fraction. Anti-nucleolin serum recog-but also has demonstrated the potential to analyze thenized a specific protein in the nucleolar fraction butcomposition of such a large subcellular organelle.not in the nucleoplasmic and cytoplasmic fractions;Moreover, the bioinformatics analysis of proteinhowever, a higher molecular weight protein could bedomain repertoire has further helped to advance thedetected in the cytoplasmic and nucleoplasmic fracunderstanding of the functional and structural diver-tion. Antifibrillarin serum recognized a specific prcsity of the nucleolus (52). The nucleoli have evolvedtein in the nucleolar fraction but not in the nucleo-from the unicellular eukaryotes to higher eukaryoticplasmic and cytoplasmic fractions. These results sug-organism. During the evolution, they have acquiredgest that the purified nucleolar preparation was highlymany new structural and functional components (7,enriched in nucleoli. Unlike human nucleoli (47, 49),53). Determining the structure and proteomic com-specific enrichment of the different isoforms of theposition of nucleoli in different species will help tonucleolin was observed in the nucleolar fraction (60.7better understand the function of nucleoli. Since bo-kDa isoform) compared to the cytoplasmic and thevine species is evolutionary divergent from humansnucleoplasmic fractions (121 kDa isoform). Theand plants, we performed a proteomic analysis of theprominent staining of the 60.7 kDa isoform in the nu-bovine nucleoli by liquid chromatography tandemcleolar fraction was indicative of the significant en-mass spectrometry (LC-MS/MS) and identified 311richment of the nucleolar fraction. The 60.7 kDa iso-proteins based on the search of available proteinform could be the alternative or processed form of nu-databases.cleolin, and its sperific 9中国煤化工TYHCNMH G146Genomics Proteomics Bioinformatics 2010 Sep; 8(3). 143-1J0Patel et al. /Proteomic Analysis of Bovine NucleolusACpERK2- 54 kDa47.7 kDa- 38 kDaNup 62- 100 kDa81 kDaNucleolin 121 kDa- 121 kDa77 kDa60.7 kDaFibrilarin42kDa -C0.5 umFigure 1 Analysis of the nucleoli isolated from MDBK cells. A. Western blot. Proteins from the cytoplasmic (ane Cp), nucleo-plasmic (lane Np) and nucleolar (lane No) fractions were separated by SDS-PAGE (10%) under reducing conditions and transferredto nitrocellulose. The separated proteins were probed with anti-ERK2, anti-Nup62, and anfifirillrin antibodies. Pre-stainedBio-Rad molecular weight markers broad range (lane M). B. Immunofluorescence. Purified nucleoli were immobilized on chamberslides (observed by DIC optics; size bar= 10 um) and immunostained with rabbit anti-fibrillarin antibody (green) and counter stainedwith pyronin Y (red). C. Electron microscopy. Left: 7,100x magnification; Right: 28,400x magnification.fraction may suggest its differential affinity for thewas highly enriched in nucleoli.nucleolar components.The integrity of the nucleoli in nucleolar prepara-To further analyze the purity of the nucleolartion was analyzed by transmission electron micros-preparations, the nucleoli were immobilized on slidescopy. As seen in Figure 1C, the purified nucleoliand analyzed by immunostaining using anti-fibrillarinmaintained the integrity and showed three distinctantibody after counter staining with the RNA bindingcompartments, that is, FC, DFC and GC.dye pyronin Y. As seen in Figure 1B, structures ob-served by the differential inference contrast (DIC)Mass spectrometry analysis of the bovine nu-optics were also stained with anti-fibrillarin serumcleoliand pyronin Y dye. These results confirmed the earlierobservation that the purified nucleolar preparationThe flowchart of the procedure used for proteomic中国煤化工MHCNMH GGenomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J8147Patel et al. / Proteomic Analysis of Bovine Nucleolusanalysis of the bovine nucleoli is shown in Figure 2A.fractionated samples were searched by Mascot data-To determine the proteomic composition of the bovinebase search using NCBInr database against all entriesnucleoli, initially, the nucleolar preparation was frac-and, by the global proteome machine (GPM) searchtionated using one-dimensional (1D) SDS-PAGE andusing Bos taurus (ENSENBL, NCBI unigene andstained with Coomassie blue. The gel was sliced intoNCBI genome), Mus musculus and Homo sapiens12 pieces and further processed by in gel-trypsin di-databases. The Mascot search identified 124 and 232gestion method (Figure 2B). Secondly, the nucleolarproteins by ID SDS-PAGE and SCX fractionation,preparation was subjected to trypsin digestion. Therespectively. Similarly, the GPM database identifiedtrypsin-digested peptides were fractionated by strong137 and 242 proteins by ID SDS-PAGE and SCXcation exchange (SCX) column and collected into 10fractionation, respectively (Figure 2C). However,fractions. Both the gel separated and the SCX fraction-comparison of the proteins identified by 1Dated samples were analyzed by RPLC ESI-MS/MS.SDS-PAGE and SCX fractionation, using both Mas-cot and GPM search, identified a total of 311 proteinsNucleolar protein identification by databasein bovine nucleoli (Table S1). Of the 311 proteins, 65searchproteins were identified by all methods, 130 proteinsidentified were common between 1D SDS-PAGE andTo identify the proteins, the mass spectra obtainedSCX fractionation, while 36 and 145 proteins werefrom the analysis of the 1D SDS-PAGE and SCXuniquely identified by each method, respectively. TheAPurified nucleoli from MDBK cellsB250 kDaSDS PAGE fractionationTrypsin Digestion150 kDa(12 fractions)3100 kDa↓4SCx fractionation- 10 fractions- 50 kDaRPLC MS/MS8- 37 kDaProtein identification by Mascot and GPM database search9Combination of idenified proteins1011. 25 kDaAnalysis by GO Miner12- 20 kDaFunctional CategorizationCSDS-PAGEsCxSDS-PAGE/GPMSCXMascotSDS-PAGE/SCX/GPMMascot29 954233199 43( 665Mascot GPMFigure 2 Mass spectrometry analysis of the bovine nucleoli. A. Flow chart of the procedure used for proteomic analysis of nucleoli.B. Proteins from the nucleolar fraction of MDBK cells were separated on 10% SDS-PAGE. C. Database search. Left: Number ofunique and common proteins identified by 1D SDS. PAGE fractionation method using Mascot and GPM database search; Middle:Number of unique and common proteins identifed by SCX fractionation method using Mascot and GPM database search; Right:Number of unique and common proteins identified by 1D SDS- PAGE and SCX methods using M ascot and GPM database search.中国煤化工MHCNMH G148Genomics Proteomics Bioinformatics 2010 Sep; 8(3). 145-1J8Patel et al. / Proteomic Analysis of Bovine NucleolusMascot and the GPM database search for the 1Dvisualized by confocal microscopy. As seen in FigureSDS-PAGE and SCX fractionated samples uniquely3, in addition to the localization largely restricted toidentified 4, 13, 21 and 31 proteins, respectivelythe nucleolus, the fusion proteins were also localized(Figure 2C). The false positive rate was analyzed byto other nuclear or cytoplasmic structures. However,reverse database search using both search engines.the results of the nucleolar localization of two fusionOnly 1 protein (0.26%) with single peptide matchingproteins (LRRC59 and AMDHD2) were not conclu-to the validation criteria was identified by Mascotsive (data not shown).human reverse database search compared to 11(2.91%) proteins identified by GPM reverse sequenceFunctional categorization of identified nucle-search (Table S2). The gene names for the proteinsolar proteinidentified from any species were assigned from theentrez gene using the gene symbols and descriptionsTo determine the functional characteristics, the identi-from their respective homologs in the human ofied nucleolar proteins were analyzed by the GoMinermouse genomes. Since the database search was per-software. The GoMiner analysis categorized a total offormed with all the entries, some peptide queries re-260, 242 and 271 proteins based on the moleculartrieved the homologous proteins from different spe-function, the biological process and the subcellularcies. Such matches were assigned the same gene namelocalization, respectively (Table 1).and their matching peptides were compiled. It is pos-In addition, the unique proteins were analyzed bysible that the homologous proteins identified by dif-pathway analysis program InnateDB (54). As seen inferent accession numbers may contain protein iso-Figure S2, while most of these proteins share a rela-forms. However, isoform analysis was not performedtively high degree of functional interconnectivity, theyin this study. The list of proteins identified along withdo not represent a simple pathway. .their gene name, accession number, species, molecu-lar weight, peptide charge, and the sequence is pro-Discussionvided in Table S2.The bovine nucleolar proteome was also blastsearched with the set of 728 proteins of human nucle-Nucleolus plays a wide variety of functions in theolar protein database. Out of 311 proteins identified incellular physiological processes (11) including thethe present study, 289 proteins had a homolog in theproduction of ribosomes, which is one of the impor-human nucleolar database, whereas 22 proteins didtant steps for the cell growth and reproduction. Be-not show significant sequence homology to any of thesides ribosome synthesis, many of the importantproteins in the human nucleolar proteome (Table S3).processes as discussed earlier are regulated by theThe fragment ion spectra of the corresponding pep-nucleolar components. Previous large-scale proteomictides identifying the 22 novel candidate proteins areanalysis of human and plant nucleoli has providedgiven in Figure SI.valuable information regarding the proteomic compo-sition and its putative role in regulating the cellularLocalization of DsRed-monomer protein fu-processes. Similarly, availability of structural and pro-sionsteomic composition of bovine nucleolus will greatlyhelp in understanding the structure of bovine nucleolusSelected cDNA clones of novel nucleolar proteinsand its role in the biology of bovine viruses includingwere individually fused inframe to DsRed-monomerbovine adenovirus type 3 (BAdV-3). As a first step to-gene in vector pDsRed-monomer N1 (Clontech) de-wards achieving our goal, we performed the proteomicsigned to express protein-DsRed monomer fusionsanalysis of bovine nucleoli for the first time.from human cytomegalovirus promoter. The VeroThe proteomic analysis of the MDBK cell nucleolicells were transfected with individual plasmid DNA.identified a total of 311 proteins. The SCX fractiona-After 48 h post transfection, the cells were stainedtion method identified higher number of proteins com-with anti-fibrillarin serum followed by DAPI andpared to the 1D SDS-PAGE fractionation; however,中国煤化工Genomics Proteomics Bioinformatics 2010 Sep; 8(3).: 14-1J8MHCNMH G149Patel et al. /Proteomic Analysis of Bovine NucleolusDsRedDAPIFibilarinMergeHMGN4C18ORF21SORBS2RSP3APRCLSSEPT5Figure 3 Subcellular localization of potential nucleolar proteins. Monolayer of Vero cells were transfected with individual plasmidDNA expressing DsRed- -monomer fusion proteins. After 48 h post-transfection, the cells were fixed and stained with rabbitanti-human fibrillarin serum followed by Cy-2 conjugated goat anti-rabbit secondary antibody. Finally the cells were incubated withDAPI and visualized by confocal microscope. Arrows indicate the nucleolar localization of DsRed-monomer tagged protein.Table 1 Categorization of the identified nucleolar proteins using GoMiner softwareMolecular functionNo. of genesBiological processCellular componentRNA binding127Ribosome biogenesis/assembly 92Nucleus214Protein binding126RNA processingCytoplasm131DNA bindingTranscription0Ribonucleoprotein)5Structural molecule activityCell cycle36NucleolusATP bindingTranslationRibosomeNTPase activity33Cell communication25ChromosomeIon binding29Stress responseMitochondrionHelicase activity23TransportCytoskeletonNuclease activity9Signal transduction20NucleoplasmTransferase activityDNA repair5snoRNP21Transporter activity7RNA splicingNucleosomeGTP binding10Chromatin assembly3ExosomeTranslation regulator activityApoptosisSpliceosomeTranscription factor activityDNA replicationCentrosomeSignal transducer activityUbiquitin cyclehnRNP中国煤化工150JMHCNMH GGenomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J0Patel et al. / Proteomic Analysis of Bovine Nucleolusboth methods uniquely identified significant numberor nucleotides may help them in the localization orof proteins thereby improving the total proteome Cov-retention of these proteins in the nucleolus. The pres-erage. The Mascot and the GPM database search fur-ence of RNA recognition motifs as predominant mo-ther improved the protein identification. Although thetifs in the human nucleolar proteins (52) may mediaterate of false positive protein identification was higherthe localization or retention of these proteins in theby the GPM search compared to the Mascot search, itnucleolus (55-63). Similarly, binding of nucleosteminwas found to be complementary to each other. How-to GTP has been shown to regulate the nucleolar lo-ever, we could not make direct comparison as bothcalzation (59, 60). Thus, binding of proteins to RNAmethods utilized different search parameters. Theor nucleotide might be one of the important mecha-number of peptides observed for each protein (Tablenisms for nucleolar targeting or retention of the pro-S1) versus observable peptides shows linear relation-teins in the nucleolus.ship with the logarithm of the protein concentrationProteins possessing catalytic activity such as(55), suggesting their relative abundance in the sam-NTPases/helicases play diverse cellular functions in-ple. A large fraction of the proteins were identifiedcluding protein folding, assembly of macromolecularbased on single or few peptide matches by either onecomplexes, organelle biogenesis, DNA replication,or both database searches. It is possible that theserecombination and repair, transcription and transportproteins are in low abundance in the nucleoli or lo-(61-63). Proteomic analysis of bovine nucleoli identi-calize under specific physiological conditions. Alter-fied significant number of proteins possessingnatively, such proteins may be the potential contami-NTPase/helicase activity, which may play essentialnants sticking to the nucleoli during purification orroles in rDNA transcription and ribosome biogenesis.part of the other subcellular component of the sameThe proteins implicated in the translational regula-density as nucleoli (56).ion (DDX48, EIF2S1, EIF2S2, EF1A1, EMG1,Among 311 proteins identified in the bovine nucle-GTF2H2, ITGBP4, and PCBP1) were also observedoli in the present study, 289 proteins (93%) showedin the bovine nucleoli. The consistent identification ofsignificant sequence homology to the proteins identi-these proteins in the human and the bovine nucleolified in the human nucleoli (44, 45, 47). Only 22 pro-supports the suggestion about the possibility of theirteins identified in the bovine nucleoli did not findinvolvement in the export of pre-assembled transla-their homolog in the human nucleolar proteome.tional machinery to the cytoplasm, mRNA surveil-These results suggest that the overall structural andlance (56, 64) or nucleolar/nuclear translation (65-67).functional organization of bovine and human nucleoliSimilarly, like human nucleoli (68), the identificationappears to be similar. Earlier report suggested thatof components involved in the ubiquitin pathwaysplant nucleoli contained a novel class of exon junction(FBXL11，POP1, RPS27A，SMURF2， UBC， andcomplex proteins not detected in the human nucleoliUSP36) in the bovine nucleoli suggest their potential(51). However, no such proteins were observed in theimplication in ribosome biogenesis.proteomic analysis of bovine nucleoli.The nucleolus has also been known to mediate the .Ribosome biogenesis is the pre-requisite process instress response by regulating the abundance of p53the formation of nucleoli. About one third of the pro-(25, 68, 69) through nucleolus -associated proteinsteins identified in the bovine nucleoli are known to beARF and MDM2 (70). Like human nucleoli (43, 44,implicated in the ribosomal gene transcription, rRNA46), we could not detect the ARF and MDM2 proteinsprocessing and ribosomal subunit assembly. A sig-in the bovine nucleoli. It is possible that their nuclearnificant number of proteins (93%) identified based onassociation may vary depending upon the physiologi-the molecular function appear to bind RNA, DNA,cal status of the cell. Alternatively, their abundancenucleotide, protein, ion, and chromatin. Since the nu-nay not be high enough to be detectable with thecleoli do not contain the membrane, their structuralmethods employed in our study. However, 10% of theintegrity could thus be maintainedproteins identified in the proteomic analysis of bovineDNA-protein, RNA-protein and protein-protein inter-nucleoli are known to be involved in the stress sig-actions. Moreover, binding of these proteins to RNAnaling (GoMin中国煤化工confirm theTYHCNMHGGenomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J8151Patel et al. / Proteomic Analysis of Bovine Nucleolusearlier observations and suggest that bovine nucleoluswell characterized (73, 74), whereas the cajal bodyplays a crucial role in regulating the cellular responsemarker protein p80-coilin was not detected in bovineto stress. In addition, the identification of significantnucleoli. However, other components known to local-number of proteins in the bovine nucleoli regulatingize in the cajal bodies, that is, fibrillarin, nucleolin,the apoptosis (4%) and cell cycle (15%) suggests thenucleolar and coiled body phosphoprotein1, nucleo-key role of bovine nucleolus in regulating these proc-phosmin, pigpen, and NHP2L1 (75-80), were detectedesses.in bovine nucleoli.The maturation and assembly of non-ribosomal ri-Of the 311 proteins identified in the purified nucle-bonucleoproteins have been known to occur in theoli by MS analysis, 22 are unique as there is no reportnucleolus (I3). A large fraction of ribonucleoproteinsin literature to support their localization in the nu-including the components of the exosome, hnRNP,cleolus. Confocal microscopy analysis of eight uniquespliceosomal, snoRNP, snRNP, RNase P and MRPproteins, each tagged with DsRed-monomer in trans-complexes were identified in the bovine nucleoli. Thefected cells, demonstrated that six are associated withexosomes are the 3'-5' exonucleases known to mediatenucleoli and other cellular structures (Figure 3).the processing of pre-rRNAs, pre-mRNAs, pre-tRNAsSimilar varied nucleolar localization patterns haveand snoRNAs (71). Although the proteomic analysisbeen observed for novel human nucleolar proteinsof the human nucleoli reported 11 subunits ofidentified by MS (47). In addition, similar estimatesexosome (46), we only identified 8 subunits in thehave been reported for nucleolar localization of pro-bovine nucleoli, including the subunits of RNase Peins as determined by analysis of MS data andand MRP complexes (POP1 and RPP30), which areYFP-tagging data (47). Absence of detection of nu-known to be involved in tRNA and rRNA processing,cleolar localization by DsRed-monomer taggingrespectively (72).analysis could be due to interference of DsRed tag inThe components of the nucleoli are highly dynamicthe fusion protein or due to less sensitivity of DsRedand their associations with the nucleoli vary depend-fluorescence compared to MS. Moreover, since nu-ing upon the physiological conditions. The gene on-cleolar localization of proteins may also vary de-tology analysis suggested that the proteins identifiedpending upon transient/stable association with nu-in the bovine nucleoli are also known to localize tocleolus, cell cycle stage, or the type of cell used (47),other cellular components such as chromosome, mi-we are confident that novel proteins identified by MStochondria, ribonucleoprotein complexes and cy-are bona fide nucleolar proteins.toskeleton. Since nucleoli organize around the ribo-The proteins uniquely identified in bovine nucleolisomal gene repeats, the identification of the chromo-(Table S3) have been shown to be involved in a vari-somal/nucleosomal proteins in the bovine nucleolarety of biological processes including metabolism, cellpreparation suggests their possible role in the organi-cycle, transcription, translation, apoptosis, ubiquitinzation of rDNA chromatin and regulation of rDNAdependent protein catabolism, and transport. Thesetranscription. Besides ribosomal proteins, the otherresults suggest that the presence of different proteinsmitochondrial proteins identified in the nucleoli couldin bovine nucleoli (or not yet identified in human nu-play essential roles in the transport of ATP and ions or .cleoli) belongs to similar functional categories.may represent potential contaminants. The proteinsknown to be the constituent of cytoskeleton were alsoidentified in our preparation, suggesting their possibleConclusionrole in the structural organization of the nucleoli orIn summary, the proteomic analysis of bovine nucleolimay also represent the potential contaminants.The nucleoli are known to play an essential role inidentified proteins that revealed significant homologythe signal recognition particle (SRP) assembly (15,(93%) to the proteins identified in human nucleoli.16). Surprisingly, the components of the SRPs wereFurthermore, our study led to the identification of 22novel candidate proteins, which do not show signifi-not detected in bovine nucleoli. Similarly, the interac-tion of cajal (coiled) body with the nucleolus has beencant sequence homology. to the proteins identified in中国煤化工MHCNMH G152Genomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J0Patel et al. / Proteomic Analysis of Bovine Nucleolushuman nucleoli. Availability of the protein data willsessed by Western blot using rabbit polyclonal anti-add the peptide information to the predicted bovinebodies recognizing cytoplasmic marker extra cellulargene sequences and would be helpful in further analy-signal-regulated kinase 2 (ERK2)， nucleoplasmicsis of the protein isoforms.marker Nucleoporin 62 (Nup62) and nucleolar mark-ers nucleolin and fibrillarin. Two micrograms of pro-tein samples from cytoplasmic, nucleoplasmic andMaterials and Methodsnucleolar fractions wereseparated or10%SDS-PAGE and electrotransferred to nitrocelluloseIsolation of nucleoli from bovine cellsmembrane. The membranes were blocked with 5%BSA and incubated with rabbit polyclonal antibodiesNucleoli from MDBK cells were isolated using aspecific to ERK2 (C-14, 1:200), Nup62 (H-122, 1:100)procedure adapted from published methods (47, 49).and fibrillarin (H-140, 1:200) (Santa Cruz) and mouseBriefly, MDBK cells were cultured at 37"C in 5% CO2anti-nucleolin monoclonal antibody (KAM-CP100,in minimum essential medium (MEM) containing 5%1:1000，Stressgen). After blocking with BSA, thefetal bovine serum (FBS). At 80% confluency, themembranes were washed three times with the TBScells were washed thrice with prewarmed phosphate(0.05 M Tris, 0.9% NaCl, pH 7.6) containing 0.1%buffered saline (PBS), trypsinized and collected inTween-20 (TBST), each for 5 min. Next, the mem-prewarmed MEM containing 5% FBS. The cells werebranes reacted with the rabbit polyclonal antisera orimmediately incubated on ice and washed thrice withthe mouse monoclonal anti-nucleolin antibody wereice-cold PBS. The protease inhibitor solution (PMSFincubated with goat anti-rabbit or goat anti-mouse1 mM, leupeptin 1 ug/mL, pepstatin 1 ug/mL, andalkaline phosphatase conjugated secondary antibody,aprotinin 1 μg/mL) was added into each buffer priorrespectively. After washing in TBST for three times (5to use. The cells were resuspended in 10 mL of hypo-nin each), the membranes were developed withtonic buffer (10 mM HEPES-KOH, pH 7.9, 1.5 mMBCIP-NBT (Sigma).MgCl2, 10 mM KCl, 0.5 mM DTT) containing 0.3%NP-40. After 30 min incubation on ice, the cells wereImmunofluorescence staining of the isolatedhomogenized using Ten Broeck homogenizernucleoli(PYREX 7727-15) until >90% of the cells burst,leaving intact nuclei with various amount of cyto-The purified nucleoli were immunostained with rabbit-plasmic material attached. Finally, the nuclei werepolyclonal anti-human firillarin specific antibodypelleted by centrifuging at 218x g for 5 min, resus-(Santa Cruz) as described earlier (47). Briefly, nucle-pended in buffer S1 (0.25 M sucrose, 10 mM MgCl2)oli were spotted on Lab-TekM II chamber slide,and centrifuged over a gradient of buffer S2 (0.35 Mair-dried, rehydrated in PBS for 5 min, and then incu-sucrose, 0.5 mM MgCl2) at 1430x g for 5 min. Thebated with anti-fibrillarin antibody (1:50). After incu-nuclear pellet thus obtained was resuspended in bufferbation for 30 min, the nucleoli were washed threeS2 and sonicated 6 times, each for 10 s on ice. Thetimes with PBS (each for 5 min) and incubated withsonicated sample was layered over a gradient 01anti-rabbit IgG (Cy-2 conjugated, 1:400) for 30 min.buffer S3 (0.88 M sucrose, 0.5 mM MgCl2) and cen-Finally, the nucleoli were counter stained with 0.66trifuged at 3000x g for 10 min. The pellet containingmM Pyronin Y (Sigma) for 1 min, washed three timesthe nucleoli was resuspended in buffer S2 and centri-with PBS (each for 5 min), mounted with DABCOfuged at 1430x g for 5 min. Finally, the nucleolar pel-containing Mowiol and observed by DIC optics andlet was resuspended in buffer S2 and stored at -70'Cfluorescent microscopy (Zeiss AxioVision).until further use.Transmission electron microscopy analysis ofWestern blot analysis of the isolated nucleoliisolated nucleoliThe enrichment of the nucleolar preparation was as-The purified nucleali weodL hy the electron中国煤化工Genomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J0TYHCNMHG153Patel et al. / Proteomic Analysis of Bovine Nucleolusmicroscopy. Briefly, the purified nucleoli were pel-earlier (8I). A total of 60 fractions were collected onleted by centrifugation at 1430x g and fixed with 3%Varian Prostar Model 330 HPLC system using a flowglutaraldehyde in 0.1 M sodium cacodylate bufferrate of 0.2 mL/min. The mobile phase solvents usedcontaining 0.35 M sucrose and 0.5 mM MgCl2:6H2O.were (1) 10 mM ammonium formiate, 25% Acetoni-The fixatives were removed and 1% agarose wastrile pH 3.0 and (2) 500 mM ammonium formiate,added to the pellet. After solidification, the pellet was25% acetonitrile, pH 6.8. After loading the sample,cut off and rinsed three times with 0.1 M sodiumisocratic conditions were maintained with 100% sol-cacodylate buffer with 0.22 M sucrose at 4C. Sam-vent A for 10 min. The peptide separation was per-ples were post fixed with 1% OsO4 in 0.1 M sodiumformed with a gradient of 0-50% solvent B for 40 mincacodylate buffer for 1 h at room temperature anfollowed by 50%- 100% solvent B for 10 min anddehydrated in 70% ethanol. After washing three times100% solvent B for 10 min. Finally, the six consecu-with propylene oxide, the samples were infiltratedtive fractions were combined to make a total of tenwith Epon/Araldite and polymerized at 55 C. Finally,fractions and analyzed by LC-MS/MS as describedthe samples were ultrathin sectioned, stained withbelow.saturated uranyl acetate and lead citrate, and viewedon Philips 410LS TEM electron microscope.LC-MS/MS1D SDS-PAGE fractionation followed by in gelThe LC_MS/MS analysis was performed using acapLC pump interfaced to a Q-Tof Ultima Global hy-digestionbrid tandem mass spectrometer fitted with a Z-sprayProteins from the nucleolar fraction were separated onnanoelectrospray ion source (Waters-Micromass). The10% SDS-PAGE, stained with Coomassie blue andsolvent A consisted of 0.2% formic acid in acetonitrilecut into 12 equal pieces. The gel pieces were placed inwhereas solvents B and C were comprised of 0.2%a 96-well microtitre plate (Sigma). The samples wereformic acid in water. The trypsin-digested samplesthen de-stained, reduced with dithiothreitol, alkylatedwere loaded onto a C18 trapping column (Sym-with iodoacetamide, and digested with porcine trypsinmetryTM 300, 0.35x5 mm Opti- pak; Waters) and(sequencing grade; Promega). The resulting peptideswashed with solvent C for 3 min at a flow rate of 30were extracted with 1% trifluroacetic acid (TFA)/2%μL/min. The flow path was switched using a 10-portacetonitrile followed by two extractions with 0.5%rotary valve before the samples were eluted onto aTFA/50% acetonitrile, and finally transferred toC18 analytical column (PepMapM, 75 um x 15 cm,96-well PCR plates using a MassPREP protein digest3-um particle size; LC Packings). The separationsstation (Micromass, Manchester, UK). The digest waswere performed using a linear gradient of 5%:95% toevaporated to dryness, resuspended in 1% aqueous40%:60% A:B in the case of gel separated proteins,TFA, and analyzed by LC-MS/MS as described be-and a linear gradient of 5%:95% to 35%:75% A:B forlow. .SCX fractionated peptides over 100 min. The compo-sition was then changed to 80%:20% A:B and held forSCX fractionation10 min to flush the column before re-equilibrating for7 min at 5%:95% A:B. Mass calibration of the Q-TofFor SCX fractionation, 200 ug of the purified nucleo-instrument was performed using a product ion spec-lar fraction was resuspended in 0.1% RapigestTM SFtrum of Glu-fibrino peptide B acquired over the m/z(Waters, Canada), reduced with dithiothreitol, alky-range of 50 to 1900. LC-MS/MS analysis was carriedlated with iodoacetamide and digested with the tryp-out using Data Dependent Acquisition, during whichsin gold (Promega) in 1:50 ratio. The tryptic digestpeptide precursor ions were detected by scanningwas acidified with 0.5% TFA and fractionated by thefrom m/z 400 to 1900 in TOF MS mode. MultiplySCX chromatography on a 200x2.1 mm Polysulphocharged (2+, 3+, or 4+) ions rising above predeter--ethyl A column (PolyLC, Columbia, USA) precededmined threshold intensity were automatically selectedby a 10x2.1 mm guard column (PolyLC) as describedfor TOF MS/MSrv collision in-中国煤化工TYHCNMH G154Genomics Proteomics Bioinformatics 2010 Sep; 8(3). 143-1J0.Patel et al. / Proteomic Analysis of Bovine Nucleolusduced dissociation of the peptides was performed insoftware (ttp://discover.nci.nih.gov/gominer/) (58),the collision cell with argon as the collision gas andand categorized based on the molecular function, bio-varying the collision energy with charge state recog-logical process and cellular component. The bovinenition. Product ion spectra were acquired over the m/znucleolar proteins were further blast searched with therange of 50 to 900. LC-MS/MS data were processeddataset of 728 proteins of human nucleolar databaseusing ProteinLynx software v4.0 (Waters-Micromass)(kindly provided by Anthony K.L. Leung, Massachu-to generate .pkl files or Mascot Distiller v2.1.0 (Ma-setts Institute of Technology, USA and Angus I. La-trixscience) to generate .mgf files.mond, University of Dundee, UK).Data analysisConstruction of DsRed monomer-nucleolarfusion proteinsThe MS/MS data files were searched against thNCBI non-redundant database for all entries usingcDNA clones containing bovine HMGN4 (8059446),Mascot v2. 1 (Matrixscience) with parameter setting as .APRC5L (8253815), RPS3 (7945675), C18ORF21follows: (1) trypsin as the specific enzyme allowing(8425238)，SorBS2 (8387106), SEPT5 (8226403),one missed cleavage, (2) peptide window tolerance ofLRRC9 (8279478) and AMDHD2 (8558625) were+0.6 Da, (3) fragment mass tolerance of +0.4 Da, (4)obtained from Open biosystems (www.openbiosystems.oxidation of methionine (fixed and variable) and (5)com). The cDNA clones and the junctions of protein-carbamidomethylation of cysteine (fixed and variable).DsRed-monomer fusions were verified by DNA se-Protein hits matching more than one peptides withquencing.individual ion score≥30 and at least one peptide withp value <0.05 or the proteins matching single peptideSubcellular localization of novel nucleolarwith an ion score≥50 and p value <0.05 were consid-proteinsered as valid protein match. Only first- rank peptideswere considered and the protein hits identified by atThe Vero cells were seeded in four-well Lab-Tekleast one unique peptide were included for furtherchamber slides. After 18 h, the cells were transfectedanalysis. The MS/MS data were further searched us-with 2 to 5 ug individual plasmid DNA using lipofec-ing the Mascot search engine against the reverse se-tamine as per manufacturer s instructions (Invitrogen).quence (NCBI human rev) database to analyze theAfter 48 h post-transfection, the cells were fixed inrate of false positive protein identifications. The data4% paraformaldehyde for 15 min, washed four timeswere also analyzed using the GPM open source soft-with PBS and permeabilized with 0.5% Triton X-100ware (57) against bovine (ENSEMBL, NCBI genome .for 20 min. The cells were stained with anti-humanand NCBI unigene database), human and mouse da-fibrillarin serum (Santa Cruz) followed by Cy-2 la-tabase using parameter setting as follows: (1) parentbeled secondary antibody. Finally， the cells wereion mass error <100 ppm, (2) fragment mass errorwashed three times with PBS, mounted in DABCO<0.4 Da, (3) trypsin as a specific enzyme allowingmounting media (Fluka) containing DAPI and ana-maximum missed cleavage of 1, (4) log(e) value <-2lyzed by confocal microscopy.(ρ value <0.01), (5) carbamido-methylation of cys-teine, and (6) oxidation of methionine along with thereversed sequence search. The GPM search also in-Acknowledgementscluded the refinement modifications， which wereoxidation and dioxidation of methionine and trypto-Authors wish to thank Dr. A.K. Leung (MIT, Cam-phan, deamidation of asparagine and glutamine, andbridge, USA) and Prof. AI. Lamond (University ofacetylation of lysine. The keratins being considered asDundee, UK) for providing human nucleolar database;potential contaminants were removed. The proteinsDr A.R.S. Ross (NRC-PBI), Dr. S. Napper (VIDO),identified by the Mascot and the GPM databaseand Dr. S. Attah Poku (VIDO) for their suggestionssearch were combined, analyzed by the GoMinerand help with中国煤化工thankful toMHCNMH GGenomics Proteomics Bioinformatics 2010 Sep; 8(3). 143-1J0.155Patel et al. /Proteomic Analysis of Bovine NucleolusMs. Sarah Caldwell (VBS, University of Saskatche-Nat. Rev. Mol. Cell Biol. 8: 574-585.wan) for electron microscopy analysis. We also thankcleoprotein maturation. Curr. Opin. Cell Biol. 15:E. Du, N. Makadiya and A. Lisanework for their help318-325.with validation of nuclear proteins. A.K. Patel was14 Filipowicz, W. and Pogacic, V.2002. Biogenesis of smallpartially supported by CGSR Devolved fellowshipnucleolar ribonucleoproteins. Curr. Opin. Cell Biol. 14:provided by University of Saskatchewan, Saskatoon,319-327.Canada. This work was supported by a grant from15 Grosshans, H, et al. 2001. Biogenesis of the signal rec-NSERC Canada to SKT.ognition particle (SRP) involves import of SRP proteinsinto the nucleolus, assembly with the SRP-RNA, andXpolp-mediated export. J. Cell Biol. 153: 745-762.Authors' contributions16 Politz, J.C, et al. 2000. Signal recognition particle com-ponents in the nucleolus. Proc. Natl. Acad. Sci. USA 97:AKP conducted the experiments, collected the data-55-60.sets，conducted data analyses, and prepared th17 Azzam, R., et al. 2004. Phosphorylation by cyclin B-Cdkmanuscript. DO and SKT supervised the project andunderlies release of mitotic exit activator Cdc 14 from theco-wrote the manuscript. All authors read and ap-nucleolus. Science 305:516-519.8 Schmidt, M.H, et al. 2003. The proliferation markerproved the manuscript.pKi-67 organizes the nucleolus during the cell cycle de-pending on Ran and cyclin B. J. Pathol. 199: 18-27.Competing interests19 Visintin, R. and Amon, A. 2000. The nucleolus: the ma-gician's hat for cell cycle tricks. Curr. Opin. Cell Biol. 12:The authors have declared that no competing interestsexist.20 Marindill, D.M. and Riley, P.R. 2008. Cell cycle switchto endocycle: the nucleolus lends a hand. Cell Cycle 7:References17-2321 Vitali, P., et al. 2005. ADAR2-mediated editing of RNAsubstrates in the nucleolus is inhibited by C/D small nu-1 Hermandez- Verdun, D.2006. The nucleolus: a model forcleolar RNAs. J. Cell Biol. 169: 745-753.the organization of nuclear functions. Histochem. Cell22 Desterro, J.M, et al. 2003. Dynamic association ofBiol.126: 135-148.RNA-editing enzymes with the nucleolus. J. Cell Sci. 116:2 Lam, Y.W., et al. 2005. The nucleolus. J. Cell Sci. 118:1805-1818.1335-1337.3 Hernandez-Verdun, D. 2006. Nucleolus: from structure tofunctional nucleolar sequestration of ADAR2. Proc. Natl.dynamics. Histochem. Cell Biol. 125: 127-137.Acad. Sci. USA 100: 14018-14023.4 Hermandez-Verdun, D., et al. 2002. Emerging concepts of24 Meder, V.S, et al. 2005. PARP-1 and PARP-2 interactnucleolar assembly. J. Cell Sci. 115: 2265-2270.with nucleophosmin/B23 and accumulate in transcrip-5 Leung, A.K. and Lamond, A.I. 2003. The dynamics oftionally active nucleoli.J. Cell Sci. 118: 211-222.Crit. Rev. Eukaryot. Gene Expr. 13: 39-54.6 Anastassova-Kristeva, M. 1977. The nucleolar cycle in25 Olson, M.O. 2004. Sensing cellular stress: another newfunction for the nucleolus? Sci. STKE 2004: pe10.man. J. Cell Sci. 25: 103-110.7 Thiry, M. and Lafontaine, D.L. 2005. Birth of a nucleolus:26 Johnson, F.B., et al. 1998. Telomeres, the nucleolus andaging. Curr. Opin. Cell Biol. 10: 332-338.the evolution of nucleolar compartments. Trends Cel27 Paushkin, S.V, et al. 2004. Identification of a humanBiol. 15: 194-199.endonuclease complex reveals a link between tRNA8 Melese, T. and Xue, Z.1995. The nucleolus: an organelleformed by the act of building a ribosome. Curr. Opin.splicing and pre-mRNA 3' end formation. Cell 117:Cell Biol.7: 319-324.9 Pederson, T. 1998. The plurifunctional nucleolus. Nucleic28 Wolin, S.L. and Matera, A.G. 1999. The trials and travelsAcids Res.26: 3871-3876.of tRNA. Genes Dev.13: 1-10.10 Lo, SJ, et al. 2006. The nucleolus: reviewing oldies to29 Sinclair, D.A., et al. 1998. Molecular mechanisms ofhave new understandings. Cell Res. 16: 530-538.yeast aging. Trends Biochem. Sci. 23: 131-134.1 Raska, I, et al. 2006. Structure and function of the nu-30 Horky, M, et al. 2002. Nucleolus and apoptosis. Ann. Ncleolus in the spotlight. Curr. Opin. Cell Biol. 18Y Acad. Sci 973: 258-264.325-334.31 Zolotukhin, A.S. and Felber, B.K. 1999. Nucleoporins2 Boisvert, F.M., et al. 2007. The multifunctional nucleolus.nup98 and nup214 participate in nuclear export of human中国煤化工MYHCNMH G156Genomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J0Patel et al. /Proteomic Analysis of Bovine Nucleolusimmunodeficiency virus type 1 Rev. J. Virol. 7352 Leung, A.K., et al. 2003. Bioinformatic analysis of thenucleolus. Biochem. J. 376: 553-569.32 Ideue, T，et al. 2004. The nucleolus is involved in53 Staub, E, et al. 2004. Insights into the evolution of themRNA export from the nucleus in fission yeast. J. Cellnucleolus by an analysis of its protein domain repertoire.Sci. 117: 2887-2895.Bioessays 26: 567-581.33 Hiscox, JA. 2002. The nucleolus- a gateway to viral in-54 Lynn， D.J， et al. 2008. InnateDB: facilitatingfection? Arch. Virol. 147: 1077-1089.systems-level analyses of the mammalian innate immune34 Hiscox, J.A. 2007. RNA viruses: hijacking the dynamicresponse. Mol. Syst. Biol. 4: 218.nucleolus. Nat. Rev. Microbiol. 5: 119-127.55 Ishihama, Y., et al. 2005. Exponentially modified protein65 Pyper, J.M., et al. 1998. The nucleolus is the site ofabundance index (emPAI) for estimation of absolute pro-Borna disease virus RNA transcription and replication. J.tein amount in proteomics by the number of sequencedVirol. 72: 7697-7702.peptides per protein. Mol. Cell Proteomics 4: 1265-1272.36 Walton, T.H, et al. 1989. Interactions of minute virus of56 Coute, Y, et al. 2006. Deciphering the human nucleolarmice and adenovirus with host nucleoli. J. Virol. 63:proteome. Mass Spectrom. Rev. 25: 215-234.365 1-3660.57 Beavis, R.C. 2006. Using the global proteome machine37 Olson, M.O. and Dundr, M. 2005. The moving parts offor protein identification. Methods Mol. Biol. 328:the nucleolus. Histochem. Cell Biol. 123: 203-216.217-228.38 Scheer, U. and Hock, R. 1999. Structure and function of58 Zeeberg, B.R., et al. 2003 GoMiner: a resource for bio-the nucleolus. Curr. Opin. Cell Biol. 11: 385-390.logical interpretation of genomic and proteomic data.39 Savino, T.M., et al. 2001. Nucleolar assembly of theGenome Biol. 4: R28.rRNA processing machinery in living cells. J. Cell Biol.Misteli, T. 2005. Going in GTP cycles in the nucleolus. J.153: 1097-1110.Ceil Biol. 168: 177-178.10 Leung, A.K., et al. 2004. Quantitative kinetic analysis of50 Tsai, R.Y. and McKay， R.D. 2005. A multistep,nucleolar breakdown and reassembly during mitosis inGTP-driven mechanism controlling the dynamic cyclinglive human cells. J. Cell Biol. 166: 787-800.of nucleostemin. J. Cell Biol. 168: 179-184.41 Gautier, T, et al. 1992. Relocation of nucleolar proteins61 Ogura, T. and Wilkinson, A.J. 2001. AAA+ superfamilyaround chromosomes at mitosis. A study by confocal la-ATPases: common structure- diverse function. Genesser scanning microscopy. J. Cell Sci. 102: 729-737.Cells 6: 575-597.42 Chen, D. and Huang, s. 2001. Nucleolar components in-52 Leipe, D.D., et al. 2004. STAND, a class of P-loopvolved in ribosome biogenesis cycle between the nucleo-NTPases including animal and plant regulators of pro-lus and nucleoplasm in interphase cells. J. Cell Biol. 153:grammed cell death: multiple, complex domain architec-169-176.tures, unusual phyletic patterns, and evolution by hori-13 Misteli, T. 2001. Protein dynamics: implications for nu-zontal gene transfer. J. Mol. Biol. 343: 1-28.clear architecture and gene expression. Science 291:63 Tanner, N.K. and Linder, P. 2001. DExD/H box RNAhelicases: from generic motors to specific dissociation14 Phair, R.D. and Misteli, T. 2000. High mobility of pro-functions. Mol. Cell 8: 251-262.teins in the mammalian cell nucleus. Nature 404:64 Wilkinson, M.F. and Shyu, A.B. 2002. RNA surveillance604-609.by nuclear scanning? Nat. Cell Biol. 4: E144-147.45 Dundr, M. and Misteli, T. 2002. Nucleolomics: an inven-65 Iborra, FJ, et al. 2001. Coupled transcription and trans-tory of the nucleolus. Mol. Cell 9: 5-7.lation within nuclei of mammalian cells. Science 293:46 Andersen, J.S, e1 al. 2005. Nucleolar proteome dynamics.1139-1142.Nature 433: 77-83.66 Iborra, FJ, et al. 2004. The case for nuclear translation. J.17 Andersen, J.S., et al. 2002. Directed proteomic analysisCell Sci. 117:5713-5720.of the human nucleolus. Curr. Biol. 12: 1-11.67 Herbert, A, et al. 2002. Induction of protein translation48 Muramatsu, M, et al. 1963. Quantitative aspects of isola-by ADARI within living cell nuclei is not dependent ontion of nucleoli of the Walker carcinosarcoma and liver ofRNA editing. Mol. Cell 10: 1235-1246.the rat. Cancer Res. 23: 510-518.58 Zhang, Y. and Xiong, Y.2001. Control of p53 ubiquiti-19 Scherl,nation and nuclear export by MDM2 and ARF. Cellhuman nucleolus. Mol. Biol. Cell 13: 4100-4109.Growth Differ. 12: 175-186.0 Busch, H, et al. 1965. Isolation of nucleoli from human59 Rubbi, C.P. and Milner, J. 2003. Disruption of the nu-cleolus mediates stabilization of p53 in response to DNA51 Pendle, A.F, et al. 2005. Proteomic analysis of thedamage and other stresses. EMBO J. 22: 6068-6077.Arabidopsis nucleolus suggests novel nucleolar functions.70 Zhang, Y. and Xiong, Y. 1999. Mutations in human ARFMol. Biol. Cell 16: 260-269.exon 2 disrr and impair its中国煤化工T.YHCNMH GGenomics Proteomics Bioinformatics 2010 Sep; 8(3).145-1J0157Patel et al. /Proteomic Analysis of Bovine Nucleolusability to block nuclear export of MDM2 and p53. Mol.77 Andrade, L.E, et al. 1991. Human autoantibody to aCell 3: 579-591.novel protein of the nuclear coiled body: immunological71 Houseley, J, et al. 2006. RNA-quality control by thecharacterization and cDNA cloning of p80-coilin. J. Exp.exosome. Nat. Rev. Mol. Cell Biol. 7: 529-539.Med.173: 1407-1419.72 Rosenblad, M.A., et al. 2006. Inventory and analysis of78 Alliegro, M.C. and Alliegro, M.A.1996. Identification ofthe protein subunits of the ribonucleases P and MRP pro-a new coiled body component. Exp. Cell Res. 227:vides further evidence of homology between the yeast386-390.and human enzymes. Nucleic Acids Res. 34: 5145-5156.79 Leung, A.K. and Lamond, A.I. 2002. In vivo analysis of73 Lyon, C.E, et al. 1997. Inhibition of protein dephos-NHPX reveals a novel nucleolar localization pathwayphorylation results in the accumulation of splicinginvolving a transient accumulation in splicing speckles. J.snRNPs and coiled bodies within the nucleolus. Exp. CellCell Biol. 157: 615-629. .Res. 230: 84-93.80 Meier, U.T. and Blobel, G. 1994. NAP57, a mammalian74 Sleeman, J,. et al. 1998. Dynamic interactions betweennucleolar protein with a putative homolog in yeast andsplicing snRNPs, coiled bodies and nucleoli revealed us-bacteria. J. Cell Biol.127: 1505-1514.ing snRNP protein fusions to the green fluorescent pro-81 Wang, H, et al. 2006. Characterization of the mousetein. Exp. Cell Res. 243: 290-304.brain proteome using global proteomic analysis comple-75 Bell, P., et al. 1992. In vitro assembly of prenucleolarmented with cysteinyl-peptide enrichment. J. Proteomebodies in Xenopus egg extract. J. Cell Biol. 118Res. 5: 361-369.1297-1304.76 Raska, I., et al. 1991. Immunological and ultrastructuralSupplementary Materialstudies of the nuclear coiled body with autoimmune an-Tables S1-S3; Figures S1 and S2tibodies. Exp. Cell Res.195: 27-37.DOI: 10.1016/S1672-0229(10)60017-4中国煤化工158Genomics Proteomics Bioinformatics 2010 Sep; 8(3.MHCNMH G