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.
Abbreviations used: CCD, charge-coupled device; CT, C-terminal glycine-rich domain; DHBB, dihydroxyboryl-Bio-Rex; DTT, dithiothreitol; GST, glutathione S-transferase; HCCA, α-cyano-4-hydroxycinnamic acid; hnRNP, heterogeneous nuclear ribonucleoprotein; MALDI-TOF MS, matrix-assisted laser-desorption–ionization time-of-flight MS; NLS, nuclear localization signal; pADPr, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; RO60, ribonucleoprotein 60; RRM, RNA recognition motif; TFA, trifluoroacetic acid.