A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins

摘要

pA 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 (9MALDI-TOF9) MS. Liquid-phase isoelectric focusing with a Rotoforsup?/sup cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotoforsup?/sup 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 sup32/supP-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. b275/b, 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./p