G2Cdb::Gene report

Gene id
G00001748
Gene symbol
PCBP1 (HGNC)
Species
Homo sapiens
Description
poly(rC) binding protein 1
Orthologue
G00000499 (Mus musculus)

Databases (7)

Gene
ENSG00000169564 (Ensembl human gene)
5093 (Entrez Gene)
886 (G2Cdb plasticity & disease)
PCBP1 (GeneCards)
Literature
601209 (OMIM)
Marker Symbol
HGNC:8647 (HGNC)
Protein Sequence
Q15365 (UniProt)

Synonyms (4)

  • HNRPE1
  • HNRPX
  • hnRNP-E1
  • hnRNP-X

Literature (43)

Pubmed - other

  • Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest.

    Waggoner SA, Johannes GJ and Liebhaber SA

    Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    The alpha-globin poly(C)-binding proteins (alphaCPs) comprise an abundant and widely expressed set of K-homolog domain RNA-binding proteins. alphaCPs regulate the expression of a number of cellular and viral mRNAs at the levels of splicing, stability, and translation. Previous surveys have identified 160 mRNAs that are bound by alphaCP in the human hematopoietic cell line, K562. To explore the functions of these alphaCP/mRNA interactions, we identified mRNAs whose levels are altered in K562 cells acutely depleted of the two major alphaCP proteins, alphaCP1 and alphaCP2. Microarray analysis identified 27 mRNAs that are down-regulated and 14 mRNAs that are up-regulated in the alphaCP1/2-co-depleted cells. This alphaCP1/2 co-depletion was also noted to inhibit cell proliferation and trigger a G(1) cell cycle arrest. Targeted analysis of genes involved in cell cycle control revealed a marked increase in p21(WAF) mRNA and protein. Analysis of mRNP complexes in K562 cells demonstrates in vivo association of p21(WAF) mRNA with alphaCP1 and alphaCP2. In vitro binding assays indicate that a 127-nucleotide region of the 3'-untranslated region of p21(WAF) interacts with both alphaCP1 and alphaCP2, and co-depletion of alphaCP1/2 results in a marked increase in p21(WAF) mRNA half-life. p21(WAF) induction and G(1) arrest in the alphaCP1/2-co-depleted cells occur in the absence of p53 and are not observed in cells depleted of the individual alphaCP isoforms. The apparent redundancy in the actions of alphaCP1 and alphaCP2 upon p21(WAF) expression correlates with a parallel redundancy in their effects on cell cycle control. These data reveal a pivotal role for alphaCP1 and alphaCP2 in a p53-independent pathway of p21(WAF) control and cell cycle progression.

    Funded by: NCI NIH HHS: P01-CA72765; NHLBI NIH HHS: HL 65449; NIDDK NIH HHS: K01 DK-071137-04

    The Journal of biological chemistry 2009;284;14;9039-49

  • Identification of transcripts and translatants targeted by overexpressed PCBP1.

    Huo LR and Zhong N

    Peking University Center of Medical Genetics, Beijing, China.

    PCBP1 is a member of the hnRNP family that functions as a RNA-binding, as well as DNA-binding, protein. The detailed transcripts and translatants targeted by PCBP1 at a global level are not yet known. We undertook an investigation of transcriptional and translational profiles after overexpressing exogenous PCBP1 in SH-SY5Y cells. Our results in two independent studies showed that 601 transcripts, including 26 down-regulated transcripts and 575 up-regulated transcripts, were impacted by overexpression of exogenous PCBP1. However, 138 and 144 transcripts showed non-overlapped differential expression in each study. These altered transcripts are clustered mainly in metabolic and transcriptional regulations. Proteomic profiles detected with a two-dimensional chromatographic PF2D showed a global change of translations, mainly in a range of pI=4.96-5.76 and pI=7.96-8.36. Three predominant proteins, which were differentially less expressed in PCBP1 overexpression cells and were detected at pI=7.96-8.16, were identified as histone proteins, indicating that histone proteins are among the targets regulated by PCBP1. Our investigation has opened a new avenue for further studying the biological functions of PCBP1.

    Biochimica et biophysica acta 2008;1784;11;1524-33

  • A cytosolic iron chaperone that delivers iron to ferritin.

    Shi H, Bencze KZ, Stemmler TL and Philpott CC

    Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

    Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human poly (rC)-binding protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.

    Funded by: Intramural NIH HHS: Z01 DK054510-03; NIDDK NIH HHS: R01 DK068139, R01 DK068139-01A1

    Science (New York, N.Y.) 2008;320;5880;1207-10

  • Pre-spliceosomal binding of U1 small nuclear ribonucleoprotein (RNP) and heterogenous nuclear RNP E1 is associated with suppression of a growth hormone receptor pseudoexon.

    Akker SA, Misra S, Aslam S, Morgan EL, Smith PJ, Khoo B and Chew SL

    Department of Endocrinology, 5th Floor, King George V Block, St Bartholomew's Hospital, West Smithfield, London EC1A 7BE, United Kingdom. s.a.akker@qmul.ac.uk

    Pseudoexons occur frequently in the human genome. This paper characterizes a pseudoexon in the GH receptor gene. Inappropriate activation of this pseudoexon causes Laron syndrome. Using in vitro splicing assays, pseudoexon silencing was shown to require a combination of a weak 5' pseudosplice-site and splicing silencing elements within the pseudoexon. Immunoprecipitation experiments showed that specific binding of heterogenous nuclear ribonucleoprotein E1 (hnRNP E1) and U1 small nuclear ribonucleoprotein (snRNP) in the pre-spliceosomal complex was associated with silencing of pseudoexon splicing. The possible role of hnRNP E1 was further supported by RNA interference experiments in cultured cells. Immunoprecipitation experiments with three other pseudoexons suggested that pre-spliceosomal binding of U1 snRNP is a potential general mechanism of suppression of pseudoexons.

    Funded by: Wellcome Trust: GR067291MA

    Molecular endocrinology (Baltimore, Md.) 2007;21;10;2529-40

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • An RNA-binding protein alphaCP-1 is involved in the STAT3-mediated suppression of NF-kappaB transcriptional activity.

    Nishinakamura H, Minoda Y, Saeki K, Koga K, Takaesu G, Onodera M, Yoshimura A and Kobayashi T

    Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

    Signal transducer and activator of transcription 3 (STAT3) has been shown to mediate the anti-inflammatory effect of IL-10. Activated STAT3 suppresses LPS-induced IL-6, tumor necrosis factor-alpha and IL-12 gene expression in macrophages and dendritic cells. However, the mechanism of Toll-like receptor (TLR) signal suppression by STAT3 has not been clarified. In this study, we investigated the effect of constitutively activated STAT3 (STAT3C) on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The forced expression of STAT3C in HEK293/TLR4 cells, but neither wild-type STAT3 nor dominant-negative form of STAT3, suppressed LPS-TLR4-mediated NF-kappaB reporter activation. The over-expression of STAT3C did not affect the signal transduction of TLR4, such as the phosphorylation of inhibitory nuclear factor-kappaBalpha and mitogen-activated protein kinases and the DNA-binding activity of NF-kappaB. Thus, STAT3C could suppress the transcriptional and/or translational activity of NF-kappaB. To define the molecular mechanism, we searched STAT3C-binding proteins by using a proteomic approach and found that a novel RNA-binding protein, alphaCP-1, interacted with STAT3C. alphaCP-1 is a K-homology domain-containing RNA-binding protein with specificity for C-rich pyrimidine tracts. Such proteins play pivotal roles in a broad-spectrum of transcriptional and translational events. The over-expression of alphaCP-1 augmented the suppressive effect of STAT3C on NF-kappaB activation in HEK293/TLR4 cells. Furthermore, the forced expression of alphaCP-1 enhanced the antagonistic effect of IL-10 on IL-6 production in RAW264.7 cells, while small interfering RNA against alphaCP-1 reduced it. These data suggest that alphaCP-1 is involved in the STAT3-mediated suppression of NF-kappaB activity.

    International immunology 2007;19;5;609-19

  • Signaling-dependent and coordinated regulation of transcription, splicing, and translation resides in a single coregulator, PCBP1.

    Meng Q, Rayala SK, Gururaj AE, Talukder AH, O'Malley BW and Kumar R

    Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

    Transcription, splicing, and translation are potentially coordinately regulatable in a temporospatial-dependent manner, although supporting experimental evidence for this notion is scarce. Yeast two-hybrid screening of a mammary gland cDNA library with human p21-activated kinase 1 (Pak1) as bait identified polyC-RNA-binding protein 1 (PCBP1), which controls translation from mRNAs containing the DICE (differentiation control element). Mitogenic stimulation of human cells phosphorylated PCBP1 on threonines 60 and 127 in a Pak1-sensitive manner. Pak1-dependent phosphorylation of PCBP1 released its binding and translational inhibition from a DICE-minigene. Overexpression of PCBP1 also inhibited the translation of the endogenous L1 cell adhesion molecule mRNA, which contains two DICE motifs in the 3' untranslated region. We also found that Pak1 activation led to an increased nuclear retention of PCBP1, recruitment to the eukaryotic translation initiation factor 4E (eIF4E) promoter, and stimulation of eIF4E expression in a Pak1-sensitive manner. Moreover, mitogenic stimulation promoted Pak1- and PCBP1-dependent alternative splicing and exon inclusion from a CD44 minigene. The alternative splicing functions of PCBP1 were in turn mediated by its intrinsic interaction with Caper alpha, a U2 snRNP auxiliary factor-related protein previously implicated in RNA splicing. These findings establish the principle that a single coregulator can function as a signal-dependent and coordinated regulator of transcription, splicing, and translation.

    Funded by: NCI NIH HHS: CA80066, CA90970, R01 CA080066, R01 CA090970

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;14;5866-71

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs.

    Kosturko LD, Maggipinto MJ, Korza G, Lee JW, Carson JH and Barbarese E

    Department of Neuroscience, Molecular, Microbial, and Structural Biology, and Biomedical Science Graduate Program, University of Connecticut Health Center, Farmington, CT 06030, USA.

    Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

    Funded by: NCRR NIH HHS: RR-02232, U54 RR022232; NINDS NIH HHS: NS-15190, NS-19943, R01 NS015190, R01 NS019943, R56 NS015190

    Molecular biology of the cell 2006;17;8;3521-33

  • A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.

    Lim J, Hao T, Shaw C, Patel AJ, Szabó G, Rual JF, Fisk CJ, Li N, Smolyar A, Hill DE, Barabási AL, Vidal M and Zoghbi HY

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

    Many human inherited neurodegenerative disorders are characterized by loss of balance due to cerebellar Purkinje cell (PC) degeneration. Although the disease-causing mutations have been identified for a number of these disorders, the normal functions of the proteins involved remain, in many cases, unknown. To gain insight into the function of proteins involved in PC degeneration, we developed an interaction network for 54 proteins involved in 23 inherited ataxias and expanded the network by incorporating literature-curated and evolutionarily conserved interactions. We identified 770 mostly novel protein-protein interactions using a stringent yeast two-hybrid screen; of 75 pairs tested, 83% of the interactions were verified in mammalian cells. Many ataxia-causing proteins share interacting partners, a subset of which have been found to modify neurodegeneration in animal models. This interactome thus provides a tool for understanding pathogenic mechanisms common for this class of neurodegenerative disorders and for identifying candidate genes for inherited ataxias.

    Funded by: NICHD NIH HHS: HD24064; NINDS NIH HHS: NS27699

    Cell 2006;125;4;801-14

  • Interaction partners for human ZNF384/CIZ/NMP4--zyxin as a mediator for p130CAS signaling?

    Janssen H and Marynen P

    Human Genome Laboratory, Department of Human Genetics, University of Leuven, Flanders Interuniversity Institute for Biotechnology (VIB), VIB4, Campus Gasthuisberg O&N 06, Herestraat 49 Box 602, B-3000 Leuven, Belgium.

    Transcription factor ZNF384/CIZ/NMP4 was first cloned in rat as a p130Cas-binding protein and has a role in bone metabolism and spermatogenesis. It is recurrently involved in translocations in acute lymphoblastic leukemia. Translocations t(12;17) and t(12;22) fuse ZNF384 to RNA-binding proteins TAF15 and EWSR1, while a translocation t(12;19) generates an E2A/ZNF384 fusion. We screened for ZNF384 interacting proteins using yeast two-hybrid technology. In contrast to its rat homolog, human ZNF384 does not interact with p130CAS. Zyxin, PCBP1, and vimentin, however, were identified as ZNF384-binding partners. Given the interaction between human zyxin and p130CAS, these results suggest that zyxin indirectly enables the interaction of ZNF384 with p130CAS which is described in rat.

    Experimental cell research 2006;312;7;1194-204

  • Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C.

    Zhong N, Radu G, Ju W and Brown WT

    Department of Human Genetics, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA.

    The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C (LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.

    Biochemical and biophysical research communications 2005;338;2;855-61

  • Transcriptome analysis of human gastric cancer.

    Oh JH, Yang JO, Hahn Y, Kim MR, Byun SS, Jeon YJ, Kim JM, Song KS, Noh SM, Yoo HS, Kim YS and Kim NS

    Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon , 305-333, Korea.

    To elucidate the genetic events associated with gastric cancer, 124,704 cDNA clones were collected from 37 human gastric cDNA libraries, including 20 full-length enriched cDNA libraries of gastric cancer cell lines and tissues from Korean patients. An analysis of the collected ESTs revealed that 97,930 high-quality ESTs coalesced into 13,001 clusters, of which 11,135 clusters (85.6%) were annotated to known ESTs. The analysis of the full-length cDNAs also revealed that 4862 clusters (51.7%) contained at least one putative full-length cDNA clone with an initiation codon, with the average length of the 5' UTR of 140 bp. A large number appear to have a diverse transcription start site (TSS). An examination of the TSS of some genes, such as TEGT and GAPD, using 5' RACE revealed that the predicted TSSs are actually found in human gastric cancer cells and that several TSSs differ depending on the specific gastric cell line. Furthermore, of the human gastric ESTs, 766 genes (9.5%) were present as putative alternatively spliced variants. Confirmation of the predicted spliced isoforms using RT-PCR showed that the predicted isoforms exist in gastric cancer cells and some isoforms coexist in gastric cell lines. These results provide potentially useful information for elucidating the molecular mechanisms associated with gastric oncogenesis.

    Mammalian genome : official journal of the International Mammalian Genome Society 2005;16;12;942-54

  • Poly(C) binding protein family is a transcription factor in mu-opioid receptor gene expression.

    Kim SS, Pandey KK, Choi HS, Kim SY, Law PY, Wei LN and Loh HH

    Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA. kimxx558@umn.edu

    The mouse mu-opioid receptor (MOR) gene has two promoters, referred to as distal and proximal promoter. Previously, our colleagues reported that a 26-base pair (bp) cis-acting element of the mouse MOR gene activates MOR gene expression. Here, we report the cloning of four members of the poly(C) binding protein (PCBP) family and show that the 26-bp polypyrimidine stretch in MOR proximal promoter interacts with these PCBPs and activates MOR transcription. The PCBPs bind not only to single-stranded but also to double-stranded DNA. The nuclear run-off assay and semiquantitative RT-PCR shows that PCBPs enhance the transcription rate of MOR gene. Furthermore, we performed refined mapping to elucidate the core region (-317/-304) involved in mediating the PCBP-induced MOR promoter activity. Decoy oligonucleotides against the polypyrimidine stretch inhibit the PCBP-induced MOR promoter activity, thereby reconfirming the role of this element in regulating MOR promoter activity. Chromatin immunoprecipitation assay confirmed the interaction of PCBPs with MOR promoter in vivo. In conclusion, we demonstrate that PCBPs act as a transcription factor and positively regulate MOR gene expression in NMB cells.

    Funded by: NIDA NIH HHS: DA00564, DA01583, DA07339, DA11190, DA11806, K02-DA13926, K05-DA00153, K05-DA070554

    Molecular pharmacology 2005;68;3;729-36

  • Nucleolar proteome dynamics.

    Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI and Mann M

    Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, Denmark.

    The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

    Funded by: Wellcome Trust: 073980

    Nature 2005;433;7021;77-83

  • RNA-binding proteins heterogeneous nuclear ribonucleoprotein A1, E1, and K are involved in post-transcriptional control of collagen I and III synthesis.

    Thiele BJ, Doller A, Kähne T, Pregla R, Hetzer R and Regitz-Zagrosek V

    Institut für Physiologie, University-Medicine Berlin, Germany.

    Collagen types I and III, coded by COL1A1/COL1A2 and COL3A1 genes, are the major fibrillar collagens produced by fibroblasts, including cardiac fibroblasts of the adult heart. Characteristic for different cardiomyopathies is a remodeling process associated with an upregulation of collagen synthesis, which leads to fibrosis. We report identification of three mRNA-binding proteins, heterogeneous nuclear ribonucleoprote (hnRNP) A1, E1, and K, as positive effectors of collagen synthesis acting at the post-transcriptional level by interaction with the 3'-untranslated regions (3'-UTRs) of COL1A1, 1A2, and 3A1 mRNAs. In vitro, binding experiments (electromobility shift assay and UV cross-linking) reveal significant differences in binding to CU- and AU-rich binding motifs. Reporter gene cell transfection experiments and RNA stability assays show that hnRNPs A1, E1, and K stimulate collagen expression by stabilizing mRNAs. Collagen synthesis is activated via the angiotensin II type 1 (AT1) receptor. We demonstrate that transforming growth factor-beta1, a major product of stimulated AT1 receptor, does not activate solely collagen synthesis but synergistically the synthesis of hnRNP A1, E1, and K as well. Thus, post-transcriptional control of collagen synthesis at the mRNA level may substantially be caused by alteration of the expression of RNA-binding proteins. The pathophysiological impact of this finding was demonstrated by screening the expression of hnRNP E1 and K in cardiovascular diseases. In the heart muscle of patients experiencing aortic stenosis, ischemic cardiomyopathy, or dilatative cardiomyopathy, a significant increase in the expression of hnRNP E1, A1, and K was found between 1.5- and 4.5-fold relative to controls.

    Circulation research 2004;95;11;1058-66

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • The WW domain-containing proteins interact with the early spliceosome and participate in pre-mRNA splicing in vivo.

    Lin KT, Lu RM and Tarn WY

    Institute of Biomedical Sciences, Academia Sinica, 128 Academy Rd., Section 2, Nankang, Taipei 11529, Taiwan.

    A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3' part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3' splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.

    Molecular and cellular biology 2004;24;20;9176-85

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • A posttranscriptional regulator of Kaposi's sarcoma-associated herpesvirus interacts with RNA-binding protein PCBP1 and controls gene expression through the IRES.

    Nishimura K, Ueda K, Guwanan E, Sakakibara S, Do E, Osaki E, Yada K, Okuno T and Yamanishi K

    Department of Microbiology, Osaka University Medical School, Suita, Osaka 565-0871, Japan.

    Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8, HHV-8) belongs to the gamma-herpesvirus subfamily. The KSHV ORF57 gene is thought to be a homolog of posttranscriptional regulators that are conserved in the herpesvirus family and are essential for replication. We generated specific monoclonal antibodies (mAbs) against the ORF57 protein that detected the 51-kDa protein expressed in the nucleus of KSHV-infected cells. We also found that the ORF57 protein interacted with poly(rC)-binding protein 1 (PCBP1), a cellular RNA-binding, posttranscriptional regulator. ORF57's interaction with PCBP1 enhanced the activity of not only poliovirus internal ribosome-entry site (IRES)-dependent translation but also X-linked inhibitor of apoptosis (XIAP) and KSHV vFLIP IRES. Actually, when ORF57 expression was induced by the expression of replication and transcription activator (RTA) in KSHV-infected cells, the expression of XIAP was enhanced. These results suggest that ORF57 binds to PCBP1 as a functional partner for posttranscriptional regulation and is involved in the regulation of the expression of both cellular and viral genes through IRESs.

    Virology 2004;325;2;364-78

  • Bag-1 internal ribosome entry segment activity is promoted by structural changes mediated by poly(rC) binding protein 1 and recruitment of polypyrimidine tract binding protein 1.

    Pickering BM, Mitchell SA, Spriggs KA, Stoneley M and Willis AE

    Department of Biochemistry, University of Leicester, LE1 7RH, United Kingdom.

    We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.

    Molecular and cellular biology 2004;24;12;5595-605

  • A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins.

    Chkheidze AN and Liebhaber SA

    Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    AlphaCPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major alphaCP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each alphaCP isoform. Immunofluorescence studies revealed three distinct distributions: alphaCP1 and alphaCP2 are predominantly nuclear with specific enrichment of alphaCP1 in nuclear speckles, alphaCP3 and alphaCP4 are restricted to the cytoplasm, and alphaCP2-KL, an alphaCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for alphaCP isoforms. alphaCP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in alphaCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in alphaCP3 or alphaCP4. Consistent with mapping studies, deletion of NLS I from alphaCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of alphaCP2. These data demonstrate an unexpected complexity in the compartmentalization of alphaCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of alphaCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.

    Funded by: NHLBI NIH HHS: R01 HL065449, R01 HL65449, R37 HL065449

    Molecular and cellular biology 2003;23;23;8405-15

  • HADHB, HuR, and CP1 bind to the distal 3'-untranslated region of human renin mRNA and differentially modulate renin expression.

    Adams DJ, Beveridge DJ, van der Weyden L, Mangs H, Leedman PJ and Morris BJ

    Basic & Clinical Genomics Laboratory, School of Medical Sciences and Institute for Biomedical Research, The University of Sydney, New South Wales 2006, Australia.

    Production of renin is critically dependent on modulation of REN mRNA stability. Here we sought to elucidate the molecular mechanisms involved. Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich "renin stability regulatory element" in the REN 3'-untranslated region (3'-UTR) contributes to basal REN mRNA instability. Yeast three-hybrid screening with the REN 3'-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) beta-subunit) as a novel REN mRNA-binding protein. Recombinant HADHB bound specifically to the 3'-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1). This required the renin stability regulatory element. Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3'-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels. Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease. Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo. Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell. Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells. In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3'-UTR of REN mRNA and regulate renin production. cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolase-dependent pathways.

    The Journal of biological chemistry 2003;278;45;44894-903

  • Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.

    Evans JR, Mitchell SA, Spriggs KA, Ostrowski J, Bomsztyk K, Ostarek D and Willis AE

    Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK.

    The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase-polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc-IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.

    Oncogene 2003;22;39;8012-20

  • Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer.

    Pillai MR, Chacko P, Kesari LA, Jayaprakash PG, Jayaram HN and Antony AC

    Department of Laboratory Medicine, Regional Cancer Centre, Thiruvananthapuram, Kerala State, India. mrpillai@vsnl.com.or

    Aims: Folate receptors (FRs) mediate cellular uptake of folates in many cancer cells and in folate deficiency heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) mediates translational upregulation of FR in cultured cervical cancer cells. hnRNP-E1 can also interfere with human papillomavirus 16 (HPV-16) viral capsid protein synthesis (and thereby HPV proliferation) in vitro. This study aimed to evaluate prospectively the relevance of FR and hnRNP-E1 expression in the normal cervix, cervical dysplasia, and cancer.

    Methods: Cervical tissues from 12 women with normal histology and 69 consecutive women with varying grades of cervical dysplasia and cancer were prospectively evaluated for immunohistochemical expression of FR, hnRNP-E1, proliferating cell nuclear antigen (PCNA), and HPV. There were 22 women with low grade squamous intraepithelial lesions (LGSIL), 22 with high grade squamous intraepithelial lesions (HGSIL), and 25 with invasive cervical carcinoma.

    Results: Among normal subjects, 100% and 92% expressed hnRNP-E1 and FR, respectively. FR expression decreased from 91% in LGSIL to 68% and 64% in women with HGSIL and cancer, respectively. Similarly, hnRNP-E1 expression decreased from 86% in LGSIL to 68% and 40% in HGSIL and cancer, respectively. There was a highly significant positive correlation between the extent of FR and hnRNP-E1 expression, and an inverse correlation between HPV infection and hnRNP-E1 expression during progression of cervical dysplasia to cancer.

    Conclusion: These results are consistent with a hypothesis that reduced hnRNP-E1 expression may be permissive for HPV proliferation and progression to cervical cancer, and support the need for prospective longitudinal studies of hnRNP-E1 expression in HPV-16 infected women.

    Funded by: NCI NIH HHS: R01 CA058919, R01CA58919; NICHD NIH HHS: R01 HD039295, R01HD39295

    Journal of clinical pathology 2003;56;8;569-74

  • The 3'-untranslated region of p21WAF1 mRNA is a composite cis-acting sequence bound by RNA-binding proteins from breast cancer cells, including HuR and poly(C)-binding protein.

    Giles KM, Daly JM, Beveridge DJ, Thomson AM, Voon DC, Furneaux HM, Jazayeri JA and Leedman PJ

    Laboratory for Cancer Medicine and University Department of Medicine, Western Australian Institute for Medical Research and Centre for Medical Research, the University of Western Australia, Perth, Western Australia 6001, Australia.

    Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.

    The Journal of biological chemistry 2003;278;5;2937-46

  • Polypyrimidine tract binding protein and poly r(C) binding protein 1 interact with the BAG-1 IRES and stimulate its activity in vitro and in vivo.

    Pickering BM, Mitchell SA, Evans JR and Willis AE

    Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.

    The 5'-untranslated region of Bag-1 mRNA contains an internal ribosome entry segment (IRES) and the translation of Bag-1 protein can be initiated by both cap-dependent and cap-independent mechanisms. In general, cellular IRESs require non-canonical trans-acting factors for their activity, however, very few of the proteins that act on cellular IRESs have been identified. Proteins that interact with viral IRESs have also been shown to stimulate the activity of cellular IRESs and therefore the ability of a range of known viral trans-acting factors to stimulate the Bag-1 IRES was tested. Two proteins, poly r(C) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB), were found to increase the activity of the Bag-1 IRES in vitro and in vivo. The regions of the Bag-1 IRES RNA to which they bind have been determined, and it was shown that PCBP1 binds to a short 66 nt section of RNA, whilst PTB interacts with a number of sites over a larger area. The minimum section of the RNA that still retained activity was determined and both PCBP1 and PTB interacted with this region suggesting that these proteins are essential for Bag-1 IRES function.

    Nucleic acids research 2003;31;2;639-46

  • Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral RNA replication.

    Walter BL, Parsley TB, Ehrenfeld E and Semler BL

    Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA.

    The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

    Funded by: NIAID NIH HHS: AI 26765, R01 AI026765, R56 AI026765

    Journal of virology 2002;76;23;12008-22

  • Comprehensive proteomic analysis of the human spliceosome.

    Zhou Z, Licklider LJ, Gygi SP and Reed R

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

    The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

    Nature 2002;419;6903;182-5

  • Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3'-untranslated region of the androgen receptor messenger RNA.

    Yeap BB, Voon DC, Vivian JP, McCulloch RK, Thomson AM, Giles KM, Czyzyk-Krzeska MF, Furneaux H, Wilce MC, Wilce JA and Leedman PJ

    Laboratory for Cancer Medicine, Western Australian Institute for Medical Research, University Department of Medicine, University of Western Australia, 50 Murray Street, Perth, WA 6000, Australia.

    The androgen receptor (AR) mediates androgen action and plays a central role in the proliferation of specific cancer cells. We demonstrated recently that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells and that androgens differentially regulate AR mRNA decay dependent on cell type (Yeap, B. B., Kreuger, R. G., Leedman, P. J. (1999) Endocrinology 140, 3282-3291). Here, we have identified a highly conserved UC-rich region in the 3-untranslated region of AR mRNA that contains a 5'-C(U)(n)C motif and a 3'-CCCUCCC poly(C)-binding protein motif. In transfection studies with LNCaP human prostate cancer cells, the AR UC-rich region reduced expression of a luciferase reporter gene. The AR UC-rich region was a target for cytoplasmic and nuclear RNA-binding proteins from human prostate and breast cancer cells as well as human testicular and breast cancer tissue. One of these proteins is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C)-binding protein-1 and -2 (CP1 and CP2), previously implicated in the control of mRNA turnover and translation, also bound avidly to the UC-rich region. Mutational analysis of the UC-rich region identified specific binding motifs for both HuR and the CPs. HuR and CP1 bound simultaneously to the UC-rich RNA and in a cooperative manner. Immunoprecipitation studies confirmed that each of these proteins associated with AR mRNA in prostate cancer cells. In summary, we have identified and characterized a novel complex of AR mRNA-binding proteins that target the highly conserved UC-rich region. The binding of HuR, CP1, and CP2 to AR mRNA suggests a role for each of these proteins in the post-transcriptional regulation of AR expression in cancer cells.

    The Journal of biological chemistry 2002;277;30;27183-92

  • Directed proteomic analysis of the human nucleolus.

    Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M and Lamond AI

    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.

    Background: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized.

    Results: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions.

    Conclusions: This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

    Current biology : CB 2002;12;1;1-11

  • Protein-protein interaction panel using mouse full-length cDNAs.

    Suzuki H, Fukunishi Y, Kagawa I, Saito R, Oda H, Endo T, Kondo S, Bono H, Okazaki Y and Hayashizaki Y

    Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, Yokohama 230-0045, Japan.

    We have developed a novel assay system for systematic analysis of protein-protein interactions (PPIs) that is characteristic of a PCR-mediated rapid sample preparation and a high-throughput assay system based on the mammalian two-hybrid method. Using gene-specific primers, we successfully constructed the assay samples by two rounds of PCR with up to 3.6 kb from the first-round PCR fragments. In the assay system, we designed all the steps to be performed by adding only samples, reagents, and cells into 384-well assay plates using two types of semiautomatic multiple dispensers. The system enabled us examine more than 20,000 assay wells per day. We detected 145 interactions in our pilot study using 3500 samples derived from mouse full-length enriched cDNAs. Analysis of the interaction data showed both several significant interaction clusters and predicted functions of a few uncharacterized proteins. In combination with our comprehensive mouse full-length cDNA clone bank covering a large part of the whole genes, our high-throughput assay system will discover many interactions to facilitate understanding of the function of uncharacterized proteins and the molecular mechanism of crucial biological processes, and also enable completion of a rough draft of the entire PPI panel in certain cell types or tissues of mouse within a short time.

    Genome research 2001;11;10;1758-65

  • Identification of two novel mammalian genes establishes a subfamily of KH-domain RNA-binding proteins.

    Makeyev AV and Liebhaber SA

    Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.

    We have identified two novel human genes encoding proteins with a high level of sequence identity to two previously characterized RNA-binding proteins, alphaCP-1 and alphaCP-2. Both of these novel genes, alphaCP-3 and alphaCP-4, are predicted to encode proteins with triplicated KH domains. The number and organization of the KH domains, their sequences, and the sequences of the contiguous regions are conserved among all four alphaCP proteins. The common evolutionary origin of these proteins is substantiated by conservation of exon-intron organization in the corresponding genes. The map positions of alphaCP-1 and alphaCP-2 (previously reported) and those of alphaCP-3 and alphaCP-4 (present report) reveal that the four alphaCP loci are dispersed in the human genome; alphaCP-3 and alphaCP-4 mapped to 21q22.3 and 3p21, and the respective mouse orthologues mapped to syntenic regions of the mouse genome, 10B5 and 9F1-F2, respectively. Two additional loci in the human genome were identified as alphaCP-2 processed pseudogenes (PCBP2P1, 21q22.3, and PCBP2P2, 8q21-q22). Although the overall levels of alphaCP-3 and alphaCP-4 mRNAs are substantially lower than those of alphaCP-1 and alphaCP-2, transcripts of alphaCP-3 and alphaCP-4 were found in all mouse tissues tested. These data establish a new subfamily of genes predicted to encode closely related KH-containing RNA-binding proteins with potential functions in posttranscriptional controls.

    Funded by: NCI NIH HHS: CA72765

    Genomics 2000;67;3;301-16

  • A set of highly conserved RNA-binding proteins, alphaCP-1 and alphaCP-2, implicated in mRNA stabilization, are coexpressed from an intronless gene and its intron-containing paralog.

    Makeyev AV, Chkheidze AN and Liebhaber SA

    Howard Hughes Medical Institute and the Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    Gene families normally expand by segmental genomic duplication and subsequent sequence divergence. Although copies of partially or fully processed mRNA transcripts are occasionally retrotransposed into the genome, they are usually nonfunctional ("processed pseudogenes"). The two major cytoplasmic poly(C)-binding proteins in mammalian cells, alphaCP-1 and alphaCP-2, are implicated in a spectrum of post-transcriptional controls. These proteins are highly similar in structure and are encoded by closely related mRNAs. Based on this close relationship, we were surprised to find that one of these proteins, alphaCP-2, was encoded by a multiexon gene, whereas the second gene, alphaCP-1, was identical to and colinear with its mRNA. The alphaCP-1 and alphaCP-2 genes were shown to be single copy and were mapped to separate chromosomes. The linkage groups encompassing each of the two loci were concordant between mice and humans. These data suggested that the alphaCP-1 gene was generated by retrotransposition of a fully processed alphaCP-2 mRNA and that this event occurred well before the mammalian radiation. The stringent structural conservation of alphaCP-1 and its ubiquitous tissue distribution suggested that the retrotransposed alphaCP-1 gene was rapidly recruited to a function critical to the cell and distinct from that of its alphaCP-2 progenitor.

    Funded by: NCI NIH HHS: CA72765-01; NHLBI NIH HHS: HL38632-6

    The Journal of biological chemistry 1999;274;35;24849-57

  • An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro.

    Wang Z, Day N, Trifillis P and Kiledjian M

    Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA.

    The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-Globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ribonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, alphaCP1 and alphaCP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have devised an in vitro mRNA decay assay which reproduces the alpha-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the alpha-complex by sequestration of alphaCP1 and alphaCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the alpha-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between alphaCP1 and alphaCP2 with PABP suggests that the alpha-complex can directly interact with PABP. Therefore, the alpha-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

    Funded by: NIDDK NIH HHS: DK51611, R01 DK051611

    Molecular and cellular biology 1999;19;7;4552-60

  • Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the alpha-globin mRNA stability complex.

    Kiledjian M, DeMaria CT, Brewer G and Novick K

    Department of Cell, Developmental and Neurobiology, Rutgers University, Piscataway, New Jersey 08855, USA. kiledjia@biology.rutgers.edu

    mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.

    Funded by: NCI NIH HHS: CA52443

    Molecular and cellular biology 1997;17;8;4870-6

  • 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.

    Blyn LB, Swiderek KM, Richards O, Stahl DC, Semler BL and Ehrenfeld E

    Department of Molecular Biology, University of California, Irvine 92717, USA.

    The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

    Funded by: NCI NIH HHS: CA33572; NIAID NIH HHS: AI12387, AI26765

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;20;11115-20

  • Assignment of human KH-box-containing genes by in situ hybridization: HNRNPK maps to 9q21.32-q21.33, PCBP1 to 2p12-p13, and PCBP2 to 12q13.12-q13.13, distal to FRA12A.

    Tommerup N and Leffers H

    Danish Center for Human Genome Research, The John F Kennedy Institute, Glostrup, Denmark. ntp@kennedy.dk.

    Genomics 1996;32;2;297-8

  • Identification of two KH domain proteins in the alpha-globin mRNP stability complex.

    Kiledjian M, Wang X and Liebhaber SA

    Howard Hughes Medical Institute, Department of Genetics, Philadelphia 19104-6145, USA.

    Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha-complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family.

    Funded by: NHLBI NIH HHS: P60-HL38632

    The EMBO journal 1995;14;17;4357-64

  • Characterisation of two major cellular poly(rC)-binding human proteins, each containing three K-homologous (KH) domains.

    Leffers H, Dejgaard K and Celis JE

    Danish Centre for Human Genome Research, Institute of Medical Biochemistry, Aarhus University, Denmark.

    We have revealed and characterised two nucleic-acid-binding proteins, termed PCBP-1 (M(r) 37,525, pI 7.07) and PCBP-2 (M(r) 38,579, pI 6.76), that together with heterogeneous ribonucleoparticle (hnRNP)-K correspond to the major cellular poly(rC)-binding proteins. mRNA for both PCBPs were detected in all the human tissues analysed. Both proteins contain three K-homologous (KH) domains which share similarity with other KH domain proteins, including the fragile-X protein FMR1, and which are positioned as in hnRNP-K and nova, i.e. with two closely spaced domains at the N-terminus and one at the C-terminus. PCBPs do not contain RGG boxes or any other known nucleic-acid-binding motifs. Expression in the vaccinia virus system showed that both proteins are post-translationally modified in vivo, a fact that was confirmed by [32P]orthophosphate labelling. Northwestern-blot analysis showed that the non-phosphorylated forms bind tenaciously to poly(rC) in vitro, while significantly less binding was observed for the phosphorylated variants. Escherichia coli expressed proteins also bound poly(rG), albeit at a lower level. In addition, PCBP-2 bound poly(rU), whereas very little binding to poly(rA) was observed for both proteins.

    European journal of biochemistry 1995;230;2;447-53

  • Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K.

    Aasheim HC, Loukianova T, Deggerdal A and Smeland EB

    Department of Immunology, Norweigian Radium Hospital, Oslo.

    In human cells at least 20 different proteins or groups of proteins have been identified that are associated with hnRNAs. These proteins (designated A1-U) are highly abundant in the nucleus. In this study, we present the sequence of a novel cDNA clone, sub2.3, isolated from a human lymphocyte cDNA library. The predicted amino acid sequence shows homology to repeated domains in the human hnRNA binding protein K (hnRNP K), which are believed to be of functional importance. hnRNP K is among the major oligo(rC/dC) binding proteins in vertebrate cells and we show here that the protein product of sub2.3 also binds to oligo(dC). This is shown by a novel approach where we demonstrated specific binding of in vitro translated sub2.3 protein to biotinylated oligo(dC) which was immobilized on magnetic streptavidin-coated Dynabeads. Moreover we found that the sub2.3 transcript is expressed in a tissue dependent manner with the highest expression observed in several lymphoid tissues and skeletal muscle. The gene was also abundantly expressed in several lymphoid cell lines and the hepatoma cell line HepG2 while a low expression was observed in the HL60 myeloid cell line and in the HeLa cervical carcinoma cell line. In conclusion, this study presents the cDNA sequence of a novel transcript which shows tissue specific expression and encodes a protein with oligo(dC) binding specificity in vitro.

    Nucleic acids research 1994;22;6;959-64

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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