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 laserdesorption-ionization time-of-flight (' MALDI-TOF ') MS. Liquid-phase isoelectric focusing with a Rotofor2 cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotofor2 protein fractions were further separated by SDS\ PAGE and then transferred to a PVDF membrane. pADPrbinding proteins were
... alysed by autoradiography of the protein blot after incubation with $#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 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, matrixassisted 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. 1 To whom correspondence should be addressed (e-mail guy.poirier!crchul.ulaval.ca). 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, . 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.