The Sound of Silence: RNAi in Poly (ADP-Ribose) Research

Blenn, Christian; Wyrsch, Philippe; Althaus, Felix R.

doi:10.3390/genes3040779

Cited by 3 publications

(3 citation statements)

References 176 publications

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“…In agreement with this observation, proliferation analysis revealed that both PARP1 KO clones showed significantly slower proliferation rates compared to WT, while the overall cell cycle distribution appeared to be unaffected (Figure 2A and B). A plethora of reports from Parp1 KO mice and human cell culture studies using RNA interference and pharmacological inhibition of PARP activity showed that loss of PARP1 leads to a sensitization of cells towards genotoxic stimuli (31,32,52). To test if the same holds true in genetically-targeted HeLa PARP1 KO cells, we performed a clonogenic survival analysis of HeLa WT and PARP1 KO cells upon H 2 O 2 treatment.…”

Section: Resultsmentioning

confidence: 99%

“…A lot of our knowledge on PARP1 and PARylation has been obtained through a series of studies using three independently generated Parp1 knock-out mouse models and immortalized mouse embryonic fibroblasts (MEFs) derived thereof (28–31), as well as siRNA-based knock-down approaches (32). Strikingly, a genetic double knock-out of Parp1 and Parp2 resulted in embryonic lethality in the mouse, thereby demonstrating a key function of PARylation during development (33).…”

Section: Introductionmentioning

confidence: 99%

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Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLaPARP1knock-out cells

et al. 2016

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Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLaPARP1knock-out cells

et al. 2016

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“…However, the PARP1 homozygous null (-/-) mouse exhibits accelerated aging and spontaneous tumorigenesis [40], and human HCT116 colon carcinoma cells that are deficient in both PARP1 alleles by CRISPR/Cas9 knockout exhibited reduced growth rates, increased cellular senescence and DNA-damage, and aberrant interferon responses [33]. These findings highlight the importance of ablating PARP1 by other less drastic methods such as partial disruption of PARP1 protein synthesis by RNAi knockdown targeting PARP1 mRNA [41] and inhibition of PARP's PARylation activity with small molecule inhibitors [42]. To understand how PARP1 modulates influenza virus life cycle, we studied the relationship between PARP1, cellular PARylation, and activity of the IAV RdRP.…”

Section: Introductionmentioning

confidence: 99%

Poly-ADP Ribosyl Polymerase 1 (PARP1) Regulates Influenza A Virus Polymerase

Westera

Jennings

Maamary

et al. 2019

Advances in Virology

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Influenza A viruses (IAV) are evolutionarily successful pathogens, capable of infecting a number of avian and mammalian species and responsible for pandemic and seasonal epidemic disease in humans. To infect new species, IAV typically must overcome a number of species barriers to entry, replication, and egress, even while virus replication is counteracted by antiviral host factors and innate immune mechanisms. A number of host factors have been found to regulate the replication of IAV by interacting with the viral RNA-dependent RNA polymerase (RdRP). The host factor PARP1, a poly-ADP ribosyl polymerase, was required for optimal functions of human, swine, and avian influenza RdRP in human 293T cells. In IAV infection, PARP1 was required for efficient synthesis of viral nucleoprotein (NP) in human lung A549 cells. Intriguingly, pharmacological inhibition of PARP1 enzymatic activity (PARylation) by 4-amino-1,8-naphthalimide led to a 4-fold increase in RdRP activity, and a 2.3-fold increase in virus titer. Exogenous expression of the natural PARylation inhibitor PARG also enhanced RdRP activity. These data suggest a virus-host interaction dynamic where PARP1 protein itself is required, but cellular PARylation has a distinct suppressive modality, on influenza A viral polymerase activity in human cells.

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