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
› InstitutsangabenFinancial 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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
53
Leissinger CA.
Platelet kinetics in immune thrombocytopenic purpura and human immunodeficiency virus thrombocytopenia. Curr Opin Hematol 2001; 08: 299-305.