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.

Alzheimer’s disease (AD) is an irreversible age-related neurodegenerative disorder clinically characterized by severe memory impairment, language deficits and cognitive decline. The major neuropathological hallmarks of AD include extracellular deposits of the β-amyloid (Aβ) peptides and cytoplasmic neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. The accumulation of plaques and tangles in the brain triggers a cascade of molecular events that culminate in neuronal damage and cell death. Despite extensive research, our understanding of the molecular basis of AD pathogenesis remains incomplete and a cure for this devastating disease is still not available. A growing body of evidence in different experimental models suggests that poly(ADP-ribose) polymerase-1 (PARP-1) overactivation might be a crucial component of the molecular network of interactions responsible for AD pathogenesis. In this work, we combined genetic, molecular and biochemical approaches to investigate the effects of two different PARP-1 inhibitors (olaparib and MC2050) in Drosophila models of Alzheimer’s disease by exploring their neuroprotective and therapeutic potential in vivo. We found that both pharmacological inhibition and genetic inactivation of PARP-1 significantly extend lifespan and improve the climbing ability of transgenic AD flies. Consistently, PARP-1 inhibitors lead to a significant decrease of Aβ42 aggregates and partially rescue the epigenetic alterations associated with AD in the brain. Interestingly, olaparib and MC2050 also suppress the AD-associated aberrant activation of transposable elements in neuronal tissues of AD flies.

Zinc finger (ZnF) proteins represent one of the largest families of human proteins, although most remain uncharacterized. Given that numerous ZnF proteins are able to interact with DNA and poly(ADP ribose), there is growing interest in understanding their mechanism of action in the maintenance of genome integrity. We now report that the ZnF protein E4F transcription factor 1 (E4F1) is an actor in DNA repair. Indeed, E4F1 is rapidly recruited, in a poly(ADP ribose) polymerase (PARP)-dependent manner, to DNA breaks and promotes ATR/CHK1 signaling, DNA-end resection, and subsequent homologous recombination. Moreover, we identify E4F1 as a regulator of the ATP-dependent chromatin remodeling SWI/SNF complex in DNA repair. E4F1 binds to the catalytic subunit BRG1/SMARCA4 and together with PARP-1 mediates its recruitment to DNA lesions. We also report that a proportion of human breast cancers show amplification and overexpression of E4F1 or BRG1 that are mutually exclusive with BRCA1/2 alterations. Together, these results reveal a function of E4F1 in the DNA damage response that orchestrates proper signaling and repair of double-strand breaks and document a molecular mechanism for its essential role in maintaining genome integrity and cell survival. Significance Cycling cells acquire numerous DNA lesions under physiological conditions and thus require functional DNA damage response (DDR) pathways to support proper DNA repair. Hundreds of proteins orchestrate the DDR in time and space from very early events, such as the sensing of DNA lesions by DNA-dependent poly(ADP-ribose) polymerases (PARPs) to late steps in chromatin restoration. Here we define a function of E4F1 in double-strand break repair. We show that E4F1 is rapidly recruited to DNA lesions by PARP-1, where it promotes γH2AX clearance, transcriptional silencing, DNA-end resection, and homologous recombination. These data provide an unsuspected molecular explanation for the essential role of E4F1 in DDR-associated cell cycle arrest and survival.

The cell cycle regulator Aurora-A kinase presents an attractive target for cancer therapies, though its inhibition is also associated with toxic side effects. To gain a more nuanced understanding of Aurora-A function, we applied shotgun proteomics to identify 407 specific protein partners, including several splicing factors. Supporting a role in alternative splicing, we found that Aurora-A localizes to nuclear speckles, the storehouse of splicing proteins. Aurora-A interacts with and phosphorylates splicing factors both in vitro and in vivo, suggesting that it regulates alternative splicing by modulating the activity of these splicing factors. Consistently, Aurora-A inhibition significantly impacts the alternative splicing of 505 genes, with RNA motif analysis revealing an enrichment for Aurora-A interacting splicing factors. Additionally, we observed a significant positive correlation between the splicing events regulated by Aurora-A and those modulated by its interacting splicing factors. An interesting example is represented by CLK1 exon 4, which appears to be regulated by Aurora-A through SRSF3. Collectively, our fi ndings highlight a broad role of Aurora-A in the regulation of alternative splicing.

Loss of von Hippel-Lindau protein pVHL function promotes VHL diseases, including sporadic and inherited clear cell Renal Cell Carcinoma (ccRCC). Mechanisms controlling pVHL function and regulation, including folding and stability, remain elusive. Here, we have identified the conserved cochaperone prefoldin complex in a screen for pVHL interactors. The prefoldin complex delivers non-native proteins to the chaperonin T-complex-protein-1-ring (TRiC) or Cytosolic Chaperonin containing TCP-1 (CCT) to assist folding of newly synthesized polypeptides. The pVHL-prefoldin interaction was confirmed in human cells and prefoldin knock-down reduced pVHL expression levels. Furthermore, when pVHL was expressed inSchizosaccharomyces pombe, all prefoldin mutants promoted its aggregation. We mapped the interaction of prefoldin with pVHL at the exon2-exon3 junction encoded region. Low levels of the PFDN3 prefoldin subunit were associated with poor survival in ccRCC patients harboring VHL mutations. Our results link the prefoldin complex with pVHL folding and this may impact VHL diseases progression.