Distinct interactomes of ADAR1 nuclear and cytoplasmic protein isoforms and their responses to interferon induction

Figures

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  Figure 1.. Interactomes of ADAR1 isoforms during homeostasis. (A) Scheme depicting the experimental approach used for the identification of stable HCIs of ADAR1. Endogenous ADAR1 and associated proteins were immunoprecipitated (IP) with an ADAR1-specific antibody and subsequently analyzed by LC–MS/MS in the DDA mode. Doxycycline-inducible stable cell lines 293 T-REx expressing either ADAR1p110 (yellow) or ADAR1p150 (green) were generated, enabling isoform-specific Strep-tag IP. The bound proteins were subjected to LC–MS/MS in both DDA and DIA modes. This image was created with BioRender. (B) Venn diagram illustrating the overlaps between the HCIs of endogenous ADAR1 and Strep-tagged ADAR1 isoforms, with both DDA and DIA modes. (C) Functional protein network of HCIs identified for both endogenous ADAR1 and Strep-tagged ADAR1 isoforms [areas highlighted in color in panel (B)]. The network was generated in STRING with medium confidence and visualized with Cytoscape. Node colors represent isoform-specific interactors (grey– both ADAR1 isoforms, yellow – ADAR1p110 unique, dark green - ADAR1p150 unique, light green - ADAR1p110 unique with DDA and both ADAR1 isoforms with DIA), outline colors…

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  Figure 2.. Distinct proximal protein networks of ADAR1 isoforms during homeostasis. (A) Scheme depicting the experimental approach used for the identification of proteins proximal to ADAR1. Doxycycline-inducible stable cell lines 293 T-REx expressing either ADAR1p110 (yellow) or ADAR1p150 (green) with BirA* at the N-terminus were generated, enabling isoform-specific proximity labeling. The biotinylated proteins bound to the beads were subjected to LC–MS/MS in the DDA mode. The image was created with BioRender. (B) Venn diagram depicting the extent of overlap between the high-confidence neighboring proteins for both ADAR1 isoforms identified with BioID. (C) Gene ontology (GO) terms of the cellular components significantly enriched for the proximal protein network of ADAR1p110 and ADAR1p150. The height of the bar corresponds to the fold enrichment and its color to the ADAR1 isoform. Small subunit (SSU) processome, cytoplasmic ribonucleoprotein granules (c. RNP granules), P-bodies (PBs), cytoplasmic SGs (c. SGs), coding region instability determinant (CRD)-mediated mRNA stability complex (CRD-complex). (D) Functional protein network of high-confidence proximal proteins identified with BioID…

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  Figure 3.. Effect of RNase A digestion on ADAR1 protein interactions. (A) Scheme depicting the experimental approach used for the identification of endogenous ADAR1 interactors occurring on long RNA. A549 cell lysates were incubated with RNase A or RNase inhibitor for 1 h at 4°C before adding either ADAR1 or IgG antibodies for subsequent overnight incubation. The image was created with BioRender. (B) Number of ADAR1 HCIs identified after SAINT filtering of co-IP samples with and without RNase A treatment. (C) The physical protein interaction network of HCIs identified in the RNase experiment and the two endogenous ADAR1 co-IP experiments. Node colors represent qualitative comparisons of bait-normalized spectral counts of HCIs between samples with and without RNase A treatment. Line thickness indicates combined Saint score, and the node size corresponds to bait-normalized spectral counts of the identified interactor.

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  Figure 4.. Effect of ADAR1 EAA mutants on protein interactions and networks. (A) Scheme depicting experimental approaches used for the identification of stable interactors and proximal protein networks occurring on dsRNA. Doxycycline-inducible stable cell lines (293 T-REx) expressing either ADAR1p110 (yellow) or ADAR1p150 (green) as well as ADAR1p110 EAA and ADAR1p150 EAA isoforms were generated. Both Strep-tag IP and BioID were performed and the bound proteins were subjected to LC–MS/MS analyses. The image was created with BioRender. (B) Number of HCIs or high-confidence proximal proteins identified with the WT and ADAR1 EAA mutants of both ADAR1 isoforms with the different methods. The color of the bar corresponds to the ADAR1 isoform (yellow– ADAR1p110 and green – ADAR1p150). (C) Dot plot of ADAR1 interactors or proximal proteins identified as showing changes with RNase A treatment that overlap in the two different experiments (Strep-tag IP and BioID) for both ADAR1 isoforms and the effect of the ADAR1 EAA mutant. The size of the dot corresponds to bait-normalized average spectra of the identified protein and color corresponds to the ADAR1 isoform (yellow– ADAR1p110 and green –…

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  Figure 5.. Comparison of interactors identified with our methods to known interactors and regulators of RNA editing. (A) Number of HCIs or proximal proteins from different datasets and the number of proteins previously described as ADAR1 interactors in two databases (BioGRID and IntACT). The height of the bar corresponds to the number of HCIs, and the filled area indicated whether they are either known or novel. (B) Heatmap showing the identification of some known proteins that interact with ADAR1 and the requirement of RNA for the interaction. Information about the RNA-dependence of the interaction was extracted from the original publication (Supplementary Table S3) and is shown on the side (N.A., not available, RNA-indep., RNA-independent, Var., variable effect, RNA-dep., RNA-dependent). The color of the tile on the heatmap corresponds to the ADAR1 isoform and the effect of RNase A digestion or ADAR1 EAA mutant (yellow– ADAR1p110 WT and EAA, light yellow – ADAR1 WT and depleted with ADAR1p110 EAA, green – ADAR1p150 and EAA, light green - depleted with ADAR1p150 EAA). Bar graphs on the right panel of the plot indicate the effect of depletion of the identified interactor on ADAR editing…

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  Figure 6.. Interactors of ADAR1 during the IFN response. (A) Scheme depicting treatments used to elucidate ADAR1 interacting protein networks during the IFN response. A549 cells were treated with either IFNα or p(I:C) for 30 h before endogenous ADAR1 co-IP. 293 T-REx cells expressing ADAR1 isoforms tagged with Strep-tag were treated with IFNα for 24 h before collection. This image was created with BioRender. (B) Number of HCIs identified as IFN-related across different treatments and datasets from this study. The height of the bar corresponds to the number of HCIs, and the gray-filled area shows those that are identified as IFN-related, with the exact number shown above the highlighted area. Proteins were annotated with the Interferome database and with GO-terms for biological processes related to IFN response. (C) Dot plot of ADAR1 interactors having log2FC (treatment over NT samples) >1 for Endo-IP, Strep-tag IP (DDA and DIA). Only HCIs identified in all three endogenous ADAR1 co-IP experiments are shown. Both the size of the point and the color gradient correspond to the log2FC of protein enriched during IFN response. (D) Dot plot showing the effect of RNase A treatment on ADAR1…

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  Figure 7.. Changes in ADAR1 proximal protein networks in the presence of both IFNα and p(I:C). (A) Scheme depicting treatments used for elucidating the proximal protein network. 293 T-REx cells were treated with IFNα or IFNα + p(I:C). Total time of treatment was 24 h. This image was created with BioRender. (B) Proximal localization of the Strep II-HA tagged ADAR1p150 isoform (green) with SGs marker – G3BP1 (red) in merged images under three conditions; NT, IFNα and IFNα + p(I:C). DAPI is shown in blue. Scale bar is 10 μm (C) Venn diagram of the overlap of high-confidence proximal proteins identified in the three conditions tested for ADAR1p110 and ADAR1p150. The color corresponds to individual treatments. (D) Dot plot of ADAR1 proximal proteins identified as unique for IFN and IFN + p(I:C) with roles in the immune response. ADAR1p110 WT and ADAR1p110 EAA mutants with different treatments are shown on the left and ADAR1p150 proteins are on the right. The size of the dot corresponds to bait-normalized average spectra of the identified protein and the color corresponds to the treatment in which the protein was identified (pink – IFNα and purple – IFNα + p(I:C)).