Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: PARylation-dependent recruitment of NCL and PABPN1 to DNA damage sites. ( A ) Live-cell imaging snapshots showing ectopic GFP-NCL and GFP-PABPN1 recruitment to laser-induced DNA damage sites in U2OS cells. Recruitment was blocked by PARP1 inhibition with PJ34. Endogenous PARP1 was detected using an RFP-fused nanobody. ( B ) Binding of NCL to PARP1 in U2OS cells was promoted by H 2 O 2 and H 2 O 2 /PARGi treatments. DNase and RNase treatment did not impact the NCL/PARP1 interaction. An anti-Flag IP was performed on lysates from Flag-PARP1 overexpressed cells, followed by immunoblotting with Flag and NCL antibodies. ( C ) Binding of PABPN1 to PARP1 in U2OS cells was promoted by H 2 O 2 and H 2 O 2 /PARGi treatment. DNase and RNase treatment did not impact the PABPN1/PARP1 interaction. An anti-Flag IP was performed on lysates from Flag-PARP1 and GFP-PABPN1 co-expressed cells, followed by immunoblotting with Flag and GFP antibodies.

Article Snippet: Stock solutions of etoposide (Sigma, Cat #E1383), PARGi (PDD 00017273, MedChemExpress, Cat #HY-108360) and PJ34 (Selleckchem, Cat #S7300) were prepared in DMSO (Dimethyl sulfoxide).

Techniques: Live Cell Imaging, Inhibition, Binding Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: The RRM of PABPN1 binds PAR and RNA competitively. ( A, B ) Pull-down analysis shows that the mutation of two aromatic residues (mR) in the RRM of PABPN1 blocks poly(A) RNA binding ( A ) but not PAR-long chain binding ( B ). ( C ) Live-cell imaging snapshots show the recruitment of ectopic GFP-PABPN1 and mutant GFP-PABPN1-mR to laser-induced DNA damage sites in U2OS cells. Recruitment was blocked by the PARP1 inhibitor PJ34. ( D ) Kinetic characterization of RNA and PAR binding to the RRMs of PABPN1 and NCL utilizing single cycle SPR. ( E ) FP assays showed that PABPN1 RRM binding to PAR and RNA is competitive in vitro . GST-RRM of PABPN1 was either incubated with poly(A) and titrated with increasing concentrations of PAR5 (PAR5 competition; black trace) or incubated with PAR5 and titrated with increasing concentrations of poly(A) (RNA competition; blue trace). ( F, G ) Pull-down assays demonstrate that PABPN1 RRM binding to PAR and RNA is competitive in vitro . GST-RRM of PABPN1 was either incubated with biotinylated PAR-long overnight, followed by incubation with varying concentrations of poly(A) for 2 h ( F ), or incubated with biotinylated poly(A) overnight, followed by incubation with varying concentrations of PAR-long for 2 h ( G ). Samples were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting with an anti-GST antibody. Poly(A) 8-mer RNA was used in all experiments.

Article Snippet: Stock solutions of etoposide (Sigma, Cat #E1383), PARGi (PDD 00017273, MedChemExpress, Cat #HY-108360) and PJ34 (Selleckchem, Cat #S7300) were prepared in DMSO (Dimethyl sulfoxide).

Techniques: Mutagenesis, RNA Binding Assay, Binding Assay, Live Cell Imaging, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot