Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Scheme of affinity purification with 2′–5′ OA conjugated beads and human, fly and mouse lysates for mass spectrometry. Cell lysates were incubated with beads linked to ATP or 2′–5′ OA, followed by affinity enrichment and analysis of bead-bound proteins using LC-MS/MS. (B-D) Identification of 2′–5′ OA binding proteins in (A) human (HeLa S3, n = 4), (B) mouse (BMDMs, n = 3) and (D) Drosophila (Schneider S2, n = 4) cell lysates. Volcano plots show the log 2 fold change enrichment of proteins in 2’–5’ OA vs ATP samples (x-axis) plotted against the log 10 transformed p-value (y-axis). Proteins in blue are significantly enriched (two-sided Student’s t-test, S0 = 0, FDR < 0.01, log 2 fold change ≥ 1.5) and proteins in orange are significantly enriched hits belonging to the ABCE and ABCF subfamilies/ABC superfamily. (E, F) Validation of ABCF1, -3 and RNAse L binding to the indicated affinity beads in HeLa (E) and THP-I (F) cell lysates that were treated with type-I IFN overnight. (G) as for (E) but 293T cell lysates from HA-ABCF1 overexpressing cells were used. (H) Binding affinity of recombinant ABCF1 to fluorescent 2′–5′ OA or OH-2′–5′ OA as determined by microscale thermophoresis (MST). The inset shows a coomassie stain on a SDS-PAGE of the recombinant protein. The graph shows mean ± sd for 2′–5′ OA (n = 4) and OH-2′–5′ OA (n = 3). (I) Precipitation of recombinant ABCF1 and the indicated variants of ABCF1 with mutations in walker A and B motifs with 2′–5′ OA and visualization by coomassie stain.

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Affinity Purification, Mass Spectrometry, Incubation, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transformation Assay, Biomarker Discovery, Recombinant, Microscale Thermophoresis, Staining, SDS Page

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Immunoblot analysis for endogenous RNase L in HeLa and HeLa S3 cells. (B, C) HeLaS3 and HeLa cells were electroporated with siRNA against ABCF1 (siABCF1) or non-targeting control siRNA (siCTRL) and expression levels of ABCF1 and GAPDH mRNA were measured by qPCR. Bar graphs show normalized average expression levels 48 h after siRNA treatment. (D, E) HeLa and HeLa S3 cells from (B, C) were transfected with 300ng polyI;C or mock transfected using Lipofectamine 2000. 24h later total RNA was isolated and visualized on an agarose gel. 28s and 18s rRNA are indicated. (F, G) HeLa S3 cells from (B) were infected with EMCV (MOI: 0.01) (F) or LACV (MOI: 1) (G) and accumulation of virus was titrated on Vero E6 cells 24h after infection. (H, I) as (F, G) but HeLa S3 cells were used. (J) A549 cells were transduced with lentiviral vectors expressing puromycin resistance, Cas9 and gRNAs targeting ABCF1, ABCF3 or non-targeting controls (NEG1 and NEG2) and selected for puromycin resistance. Obtained cells were infected with the indicated viruses expressing fluorescent reporter proteins. The bar graphs show mean fluorescent intensity (± sd, n=4) normalized to cell confluence for timepoint of 24 hpi (VSV-eGFP MOI 0.2, RVFV-Katushka MOI 0.5), 40 hpi (YFV-Venus MOI 2), 48 hpi (HSV-1-mCherry MOI 2) or 72 hpi (VACV-eGFP MOI 0.01). One representative experiment of three is shown.

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Western Blot, Control, Expressing, Transfection, Isolation, Agarose Gel Electrophoresis, Infection, Virus, Transduction

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Experimental scheme of the pulsed SILAC approach used to measure protein synthesis in HeLa and HeLa S3 cells after treatment with OH-2′–5′ OA or 2′–5′ OA. (B) Density scatter plot comparing the normalized and log 2 transformed intensity of heavy SILAC-labelled proteins in HeLa S3 cells treated with 2′–5′ OA or OH-of 2′–5′ OA (n = 4). Diagonal lines indicate a slope of 1, including an offset of ± log 2 0.5 in case of dashed lines. (C) As (B) in HeLa cells with low RNase L levels. (D, E) MEFs were treated with siRNA for ABCF1 or non-targeting control for 48 h and treated with the indicated stimuli. (D) Abundance of ABCF1 normalized to GAPDH transcripts in relation to mock siCTRL. Graph shows mean (± sd, n = 2) of one representative experiment of two. (E) MEF cells were treated with different stimuli to induce type-I interferon production. After 24 h the supernatants were assayed for presence of typ-I interferon using a bioassay on ISRE-Luc reporter containing L292 cell line. Gaph shows mean (± sd, n = 3) of one representative experiment of three.

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Multiplex sample analysis, Transformation Assay, Control, Bioassay

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Comparison of fraction of 2′–5′ OA bound with or without the 5′ triphosphate. Calf-intestinal phosphatase treatment removes the 5′ triphosphate and reduces mDecr1-2′–5′ OA bound complex formation. (B) 1.35 Å crystal structure of mouse Decr1 bound to 2′–5′ OA. Decr1 is a homo-tetrameric metabolic auxiliary enzyme that catalyzes the reduction of trans-unsaturated fatty acids in the mitochondria. We see electron density for one molecule of 2′–5′ OA with three clearly defined bases bound to each monomer of Decr1. (C) 2Fo-Fc map of 2′–5′ OA bound to one monomer of Decr1 contoured to 1 σ. (D) 2′–5′ OA bound to mouse Decr1. 2′–5′ OA contacts charged residues in each Decr1 monomer with specific contacts to the 2′ phosphate and first base. Binding pocket and active site residues in mDecr1 and hDecr1 are highly conserved (compare to E). (E) NADPH bound to human Decr1 (PDB: 1W6U) with 2′–5′ OA modeled and overlayed. 2′–5′ OA binds in an extended binding site compared to NADPH, which has key contacts deeper in the binding pocket. N92 in mDecr1 is permissive to 2′–5′ OA binding, while K92 in hDecr1 allows NADPH binding but sterically clashes with modelled 2′–5′ OA binding. (F) Comparison of mouse Decr1, human Decr1, or human mutant K92N and 2′–5′ OA complex formation. WT human Decr1 cannot form a complex with 2′–5′ OA, while the K92N mutation in the human Decr1 background is permissive to 2′–5′ OA binding and complex formation. (G) NIH-3T3 cells were transduced with lentiviral vectors expressing Cas9 and gRNA targeting Abcf1, Abcf3, Decr1, Mavs or non-targeting control (Neg). Targeted cell populations after puromycin selection were infected with viruses encoding fluorescent reporter genes. The graphs show mean fluorescent intensity (± sd, n = 4) normalized to cell confluence for timepoints 27 hpi (VSV-eGFP MOI 0.1), 48 hpi (RVFV-Katushka MOI 0.1), 60 hpi (YFV-Venus MOI 0.5), 72 hpi (HSV-1-eGFP MOI 1) or 96 hpi (VACV-eGFP MOI 0.01).

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Comparison, Binding Assay, Mutagenesis, Transduction, Expressing, Control, Selection, Infection