A new class of poly(ADP-ribose) (pADPr)-binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), has been identified by a proteomic approach using matrix-assisted laser-desorption–ionization time-of-flight ('MALDI-TOF') MS. Liquid-phase isoelectric focusing with a Rotofor® cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotofor® protein fractions were further separated by SDS/PAGE and then transferred to a PVDF membrane. pADPr-binding proteins were analysed by autoradiography of the protein blot after incubation with 32P-labelled automodified pADPr polymerase-1 (PARP-1). Peptide mass fingerprinting of selected bands identified the most abundant pADPr-binding proteins as hnRNPs, a family of proteins that bind pre-mRNA into functional complexes involved in mRNA maturation and transport to the cytoplasm. Sequence homology database searching against a previously reported pADPr-binding sequence motif revealed that the hnRNPs contain a putative pADPr-binding sequence pattern [Pleschke, Kleczkowska, Strohm and Althaus (2000) J. Biol. Chem. 275, 40974–40980]. pADPr-binding assays performed with synthetic peptides by the dot-blot technique and with nitrocellulose-transferred recombinant hnRNPs confirmed the pADPr-binding protein identification and the specificity of the interaction. These results could establish a link between increased levels of pADPr in DNA damaged cells and the modified protein expression pattern resulting from altered mRNA trafficking.
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April 2003
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Research Article|
April 15 2003
A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins
Jean-Philippe GAGNÉ;
Jean-Philippe GAGNÉ
∗Health and Environment Unit, Laval University Medical Research Center, Centre hospitalier universitaire de Québec (CHUQ), Faculty of Medicine, Laval University, 2705, Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2
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Joanna M. HUNTER;
Joanna M. HUNTER
∗Health and Environment Unit, Laval University Medical Research Center, Centre hospitalier universitaire de Québec (CHUQ), Faculty of Medicine, Laval University, 2705, Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2
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Benoît LABRECQUE;
Benoît LABRECQUE
†Department of Microbiology and Infectiology, Faculty of Medicine, Sherbrooke University, 3001, North 12th Avenue, Fleurimont, Québec, Canada J1H 5N4
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Benoît CHABOT;
Benoît CHABOT
†Department of Microbiology and Infectiology, Faculty of Medicine, Sherbrooke University, 3001, North 12th Avenue, Fleurimont, Québec, Canada J1H 5N4
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Guy G. POIRIER
Guy G. POIRIER
1
∗Health and Environment Unit, Laval University Medical Research Center, Centre hospitalier universitaire de Québec (CHUQ), Faculty of Medicine, Laval University, 2705, Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2
1To whom correspondence should be addressed (e-mail guy.poirier@crchul.ulaval.ca).
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Publisher: Portland Press Ltd
Received:
October 25 2002
Revision Received:
December 17 2002
Accepted:
January 08 2003
Accepted Manuscript online:
January 08 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 371 (2): 331–340.
Article history
Received:
October 25 2002
Revision Received:
December 17 2002
Accepted:
January 08 2003
Accepted Manuscript online:
January 08 2003
Citation
Jean-Philippe GAGNÉ, Joanna M. HUNTER, Benoît LABRECQUE, Benoît CHABOT, Guy G. POIRIER; A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins. Biochem J 15 April 2003; 371 (2): 331–340. doi: https://doi.org/10.1042/bj20021675
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