Fig. 5: A3C proteins predominantly bind the N-terminal region of DDX5.

A A3C pulldown and immunoblot analysis of H1299 cell lysates with or without gemcitabine treatment. * Shorter exposure time. B Venn diagram illustrated the intersection of A3C interactome with or without gemcitabine treatment. C Enrichment analysis of pathways based on Gene Ontology (GO). D A3C interactome was screened by mass spectrometry-based proteomics. The table showed the representative highest scored peptides that physically bind to A3C. Our AP-MS dataset was verified by published proteomic data (Cheung group, 2018) from RNA/DNA hybrids immunoprecipitation (DRIP) located at the BAMBI promoter and DPP9 3’ UTR. E A Venn diagram illustrated the intersection of A3C interactome (291, after removed keratins and other contaminants) with 803 proteins that interact with RNA/DNA hybrids published by Cheung group. Molecular Complex Detection (MCODE) was executed to identify key modules in the protein-protein interaction (PPI) network of A3C-hybrids binding. DDX5, DDX3X and DHX9 were highlighted in the highly interconnected clusters. F R-loop-dependent A3C-DDX5 interaction. H1299 cells grown on 10 cm dishes were co-transfected with 5 μg FLAG-tagged A3C and 5 μg HA-tagged DDX5, along with either 5 μg wild-type RNase H1 (ppyCAG_RNaseH1), the catalytically inactive D210N mutant, or an empty vector (pcDNA3.1). Cell lysates were analyzed by immunoprecipitation using anti-FLAG antibodies followed by immunoblots with the indicated antibodies. Input indicates 20% of pre-immunoprecipitated samples. G The full-length (FL), N-terminal (ΔΝ), C-terminal (ΔC) or both (ΔΝC) truncated forms of DDX5. H HEK-H293T cells were transfected with full-length (FL) HA-DDX5, or the DDX5 truncation mutants for 24 h. Lysates were incubated with immobilized HA. Affinity precipitated exogenous DDX5 was detected with anti-HA antibody, with the relative levels in the lysates also shown. I Display of the Predicted Aligned Error (PAE) for the AlphaFold-predicted A3C/DDX5 complex. DDX5 and A3C chains are labeled respectively in cyan and orange. A dark green tile designates a good prediction, and the region of DDX5 that is expected to be in proximity to A3C includes residues 50-100, while for A3C, the residue range is 30-180. Structure-based protein interaction interface analysis between A3C and DDX5. Image represents the predicted A3C-DDX5 complex, where interaction interface and hotspot residues are labeled. J Structure-based protein interaction interface between A3C (PDB ID: 3vow) and DDX5 (PDB ID: 4a4d) where interaction region and two hotspot residues (Tyr59/Tyr97) are labeled.