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Thromb Haemost 2006; 95(04): 643-651
DOI: 10.1160/TH05-11-0764
Platelets and Blood Cells
Schattauer GmbH

Analysis of SAGE data in human platelets: Features of the transcriptome in an anucleate cell

Marcus Dittrich
1   Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg
2   Institut für Klinische Biochemie und Pathobiochemie, Würzburg
,
Ingvild Birschmann
2   Institut für Klinische Biochemie und Pathobiochemie, Würzburg
,
Julia Pfrang
2   Institut für Klinische Biochemie und Pathobiochemie, Würzburg
,
Sabine Herterich
2   Institut für Klinische Biochemie und Pathobiochemie, Würzburg
,
Albert Smolenski
4   Institute of Biochemistry II, Medical Faculty, University of Frankfurt, Frankfurt; Germany
,
Ulrich Walter
2   Institut für Klinische Biochemie und Pathobiochemie, Würzburg
,
Thomas Dandekar
1   Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg
3   EMBL, Heidelberg
› Author AffiliationsFinancial support: The study was supported by the IZKF Würzburg (MD / PhD program), the Deutsche Vereinigte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin (DGKL) and the DFG (SPP grant Bo 1099/5–3; SFB 688/TP A2).
Further Information

Publication History

Received 24 November 2005

Accepted after revision 19 February 2006

Publication Date:
30 November 2017 (online)

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Summary

A comprehensive SAGE (serial analysis of gene expression) library of purified human platelets was established. Twenty-five thousand (25,000) tags were sequenced, and after removal of mitochondrial tags, 12,609 (51%) non-mitochondrial-derived tags remained, corresponding to 2,300 different transcripts with expression levels of up to 30,000 tags per million. This new, highly purified SAGE library of platelets is enriched in specific transcripts.The complexity in terms of tag distribution is similar to cells that are still able to replenish their mRNA pool by transcription.We show that our SAGE data are consistent with recently published microarray data but show further details of the platelet transcriptome, including (i) longer UTR regions and more stable folding in the enriched mRNAs, (ii) biologically interesting new candidate mRNAs that show regulatory elements, including elements for RNA stabilization or for translational control, and (iii) significant enrichment of specific, highly transcribed mRNAs compared to a battery of SAGE libraries from other tissues. Among several regulatory mRNA elements known to be involved in mRNA localization and translational control, CPE elements are in particular enriched in the platelet transcriptome. mRNAs previously reported to be translationally regulated were found to be present in the library and were validated by real-time PCR. Furthermore, specific molecular functions such as signal transduction activity were found to be significantly enriched in the platelet transcriptome.These findings emphasize the richness and diversity of the platelet transcriptome.

Keywords

Platelet - transcriptome - SAGE - signaling pathway - post-transcriptional regulation - RNA element - gene ontology

 

  • References

  • 1 Ts’ao CH. Rough endoplasmic reticulum and ribosomes in blood platelets. Scand J Haematol 1971; 08: 134-40.
    PubMedGoogle Scholar
  • 2 McRedmond JP, Park SD, Reilly DF. et al. Integration of proteomics and genomics in platelets:a profile of platelet proteins and platelet-specific genes. Mol Cell Proteomics 2004; 03: 133-44.
    CrossrefPubMedGoogle Scholar
  • 3 Gnatenko DV, Dunn JJ, McCorkle SR. et al. Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 2003; 101: 2285-93.
    CrossrefPubMedGoogle Scholar
  • 4 Booyse FM, Rafelson Jr ME. Stable messenger RNA in the synthesis of contractile protein in human platelets. Biochim Biophys Acta 1967; 145: 188-90.
    CrossrefPubMedGoogle Scholar
  • 5 Kieffer N, Guichard J, Farcet JP. et al. Biosynthesis of major platelet proteins in human blood platelets. Eur J Biochem 1987; 164: 189-95.
    CrossrefPubMedGoogle Scholar
  • 6 Weyrich AS, Dixon DA, Pabla R. et al. Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets. Proc Natl Acad Sci USA 1998; 95: 5556-61.
    CrossrefPubMedGoogle Scholar
  • 7 Pabla R, Weyrich AS, Dixon DA. et al. Integrin-dependent control of translation: engagement of integrin αIIbβ3 regulates synthesis of proteins in activated human platelets. J Cell Biol 1999; 144: 175-84.
    CrossrefPubMedGoogle Scholar
  • 8 Lindemann S, Tolley ND, Dixon DA. et al. Activated platelets mediate inflammatory signaling by regulated interleukin 1β synthesis. J Cell Biol 2001; 154: 485-90.
    CrossrefPubMedGoogle Scholar
  • 9 Rosenwald IB, Pechet L, Han A. et al. Expression of translation initiation factors elF-4E and elF-2α and a potential physiologic role of continuous protein synthesis in human platelets. Thromb Haemost 2001; 85: 142-51.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 10 Booyse FM, Rafelson Jr ME. Studies on human platelets. I. Synthesis of platelet protein in a cell-free system. Biochim Biophys Acta 1968; 166: 689-97.
    CrossrefPubMedGoogle Scholar
  • 11 Booyse FM, Hoveke TP, Rafelson Jr ME. Studies on human platelets. II. Protein synthetic activity of various platelet populations. Biochim Biophys Acta 1968; 157: 660-3.
    CrossrefPubMedGoogle Scholar
  • 12 Santoso S, Kalb R, Kiefel V. et al. The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis. Br J Haematol 1993; 84: 451-6.
    CrossrefPubMedGoogle Scholar
  • 13 Booyse F, Rafelson Jr ME. In vitro incorporation of amino-acids into the contractile protein of human blood platelets. Nature 1967; 215: 283-4.
    CrossrefPubMedGoogle Scholar
  • 14 Lindemann S, Tolley ND, Eyre JR. et al. Integrins regulate the intracellular distribution of eukaryotic initiation factor 4E in platelets. A checkpoint for translational control. J Biol Chem 2001; 276: 33947-51.
    CrossrefPubMedGoogle Scholar
  • 15 Bessler H, Agam G, Djaldetti M. Increased protein synthesis by human platelets during phagocytosis of latex particles in vitro . Thromb Haemost 1976; 35: 350-7.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 Plow EF. Extracellular factors influencing the in vitro protein synthesis of platelets. Thromb Haemost 1979; 42: 666-78.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Denis MM, Tolley ND, Bunting M. et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005; 122: 379-91.
    CrossrefPubMedGoogle Scholar
  • 18 Watson SP, Bahou WF, Fitzgerald D. et al. Mapping the platelet proteome: a report of the ISTH Platelet Physiology Subcommittee. J Thromb Haemost 2005; 03: 2098-101.
    CrossrefPubMedGoogle Scholar
  • 19 Dittrich M, Birschmann I, Stuhlfelder C. et al. Understanding platelets. Lessons from proteomics, genomics and promises from network analysis. Thromb Haemost 2005; 94: 916-25.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 20 Velculescu VE, Zhang L, Vogelstein B. et al. Serial analysis of gene expression. Science 1995; 270: 484-7.
    CrossrefPubMedGoogle Scholar
  • 21 van der Meer PF, Gratama JW, van Delden CJ. et al. Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components. Br J Haematol 2001; 115: 953-62.
    CrossrefPubMedGoogle Scholar
  • 22 Smolenski A, Schultess J, Danielewski O. et al. Quantitative analysis of the cardiac fibroblast transcriptome-implications for NO/cGMP signaling. Genomics 2004; 83: 577-87.
    CrossrefPubMedGoogle Scholar
  • 23 Wittwer CT, Ririe KM, Andrew RV. et al. The Light-Cycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 1997; 22: 176-81.
    CrossrefPubMedGoogle Scholar
  • 24 Lash AE, Tolstoshev CM, Wagner L. et al. SAGEmap: a public gene expression resource. Genome Res 2000; 10: 1051-60.
    CrossrefPubMedGoogle Scholar
  • 25 Harris MA, Clark J, Ireland A. et al. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res 2004; 32 Database issue: D258-61.
    CrossrefPubMedGoogle Scholar
  • 26 Hofacker IL. Vienna RNA secondary structure server. Nucleic Acids Res 2003; 31: 3429-31.
    CrossrefPubMedGoogle Scholar
  • 27 Pesole G, Liuni S. Internet resources for the functional analysis of 5’ and 3’ untranslated regions of eukaryotic mRNAs. Trends Genet 1999; 15: 378.
    CrossrefPubMedGoogle Scholar
  • 28 Bengert P, Dandekar T. A software tool-box for analysis of regulatory RNA elements. Nucleic Acids Res 2003; 31: 3441-5.
    CrossrefPubMedGoogle Scholar
  • 29 Dijkstra-Tiekstra MJ, van der Meer PF, Pietersz RN. et al. Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells. Transfusion 2004; 44: 1319-24.
    CrossrefPubMedGoogle Scholar
  • 30 Mazumder B, Seshadri V, Fox PL. Translational control by the 3’-UTR: the ends specify the means. Trends Biochem Sci 2003; 28: 91-8.
    CrossrefPubMedGoogle Scholar
  • 31 Mendez R, Richter JD. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2001; 02: 521-9.
    PubMedGoogle Scholar
  • 32 Barnard DC, Ryan K, Manley JL. et al. Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation. Cell 2004; 119: 641-51.
    CrossrefPubMedGoogle Scholar
  • 33 Lai EC. Micro RNAs are complementary to 3’ UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 2002; 30: 363-4.
    CrossrefPubMedGoogle Scholar
  • 34 Chen CZ, Li L, Lodish HF. et al. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303: 83-6.
    CrossrefPubMedGoogle Scholar
  • 35 Lewis BP, Shih IH, Jones-Rhoades MW. et al. Prediction of mammalian microRNA targets. Cell 2003; 115: 787-98.
    CrossrefPubMedGoogle Scholar
  • 36 Gagnon AW, Murray DL, Leadley RJ. Cloning and characterization of a novel regulator of G protein signalling in human platelets. Cell Signal 2002; 14: 595-606.
    CrossrefPubMedGoogle Scholar
  • 37 Garcia A, Prabhakar S, Hughan S. et al. Differential proteome analysis of TRAP-activated platelets: Involvement of DOK-2 and phosphorylation of RGS proteins. Blood 2004; 103: 2088-95.
    CrossrefPubMedGoogle Scholar
  • 38 Mineo C, Ying YS, Chapline C. et al. Targeting of protein kinase Cγ to caveolae. J Cell Biol 1998; 141: 601-10.
    CrossrefPubMedGoogle Scholar
  • 39 Saha S, Sparks AB, Rago C. et al. Using the transcriptome to annotate the genome. Nat Biotechnol 2002; 20: 508-12.
    CrossrefPubMedGoogle Scholar
  • 40 Paillard L, Osborne HB. East of EDEN was a poly(A) tail. Biol Cell 2003; 95: 211-9.
    CrossrefPubMedGoogle Scholar
  • 41 Sachs AB, Sarnow P, Hentze MW. Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell 1997; 89: 831-8.
    CrossrefPubMedGoogle Scholar
  • 42 Neininger A, Kontoyiannis D, Kotlyarov A. et al. MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels. J Biol Chem 2002; 277: 3065-8.
    CrossrefPubMedGoogle Scholar
  • 43 Ostareck-Lederer A, Ostareck DH, Hentze MW. Cytoplasmic regulatory functions of the KH-domain proteins hnRNPs K and E1/E2. Trends Biochem Sci 1998; 23: 409-11.
    CrossrefPubMedGoogle Scholar
  • 44 Cao Q, Richter JD. Dissolution of the maskineIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation. Embo J 2002; 21: 3852-62.
    CrossrefPubMedGoogle Scholar
  • 45 Atkins CM, Nozaki N, Shigeri Y. et al. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulindependent protein kinase II. J Neurosci 2004; 24: 5193-201.
    CrossrefPubMedGoogle Scholar
  • 46 Gardiner EE, Arthur JF, Kahn ML. et al. Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase. Blood 2004; 104: 3611-7.
    CrossrefPubMedGoogle Scholar
  • 47 Rabie T, Strehl A, Ludwig A. et al. Evidence for a role of ADAM17 (TACE) in the regulation of platelet glycoprotein V. J Biol Chem 2005; 280: 14462-8.
    CrossrefPubMedGoogle Scholar
  • 48 Diamant M, Tushuizen ME, Sturk A. et al. Cellular microparticles: new players in the field of vascular disease?. Eur J Clin Invest 2004; 34: 392-401.
    CrossrefPubMedGoogle Scholar
  • 49 Marcus K, Immler D, Sternberger J. et al. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis 2000; 21: 2622-36.
    CrossrefPubMedGoogle Scholar
  • 50 O’Neill EE, Brock CJ, von Kriegsheim AF. et al. Towards complete analysis of the platelet proteome. Proteomics 2002; 02: 288-305.
    CrossrefPubMedGoogle Scholar
  • 51 Garcia A, Prabhakar S, Brock CJ. et al. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2004; 04: 656-68.
    CrossrefPubMedGoogle Scholar
  • 52 Moebius J, Zahedi RP, Lewandrowski UU. et al. The human platelet membrane proteome reveals several new potential membrane proteins. Mol Cell Proteomics 2005; 04: 1754-61.
    CrossrefPubMedGoogle Scholar
  • 53 Leissinger CA. Platelet kinetics in immune thrombocytopenic purpura and human immunodeficiency virus thrombocytopenia. Curr Opin Hematol 2001; 08: 299-305.
    CrossrefPubMedGoogle Scholar
  • 54 Hillmann AG, Harmon S, Park SD. et al. Comparative RNA expression analyses from small-scale, single-donor platelet samples. J Thromb Haemost 2006; 04: 349-56.
    CrossrefPubMedGoogle Scholar