A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins.
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 (32)P-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.
Gagné
Jean-Philippe
JP
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.
Hunter
Joanna M
JM
Labrecque
Benoît
B
Chabot
Benoît
B
Poirier
Guy G
GG
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011064:Poly Adenosine Diphosphate Ribose / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; C024857:poly(ADP)-ribosylated proteins; D011064:Poly Adenosine Diphosphate Ribose