Extended Data Fig. 10: (Related to Fig. 5). Synthetic host cell-derived Z-RNAs and pharmacological CPSF3 inhibition activate ZBP1.
From: Host cell Z-RNAs activate ZBP1 during virus infections
a, Schematic of synthetic RNA hairpins generated from predicted virus-induced host Z-RNAs. The coverage track displays the Z22 RIP signal from infected cells, with red ticks indicating REDIportal-annotated or newly identified A → I editing sites. Synthetic hairpins were generated from predicted Z-RNA stems within Z22 peaks (orange highlight) formed by pairs of inverted LINE (Vbp1), LTR (Hmga1), and SINE (Ptbp1) elements. The minimum free energy structure of each hairpin is shown at the bottom, with the artificial outer UUUC loop rendered semitransparent. 2′-O-methyl-8-methylguanosine (m8Gm) modified bases are circled in red. b, Immunofluorescence staining of Z-RNA and FAM in primary MEFs transfected with RNA hairpins designed from putative Z-forming sequences withing aberrant host cell transcripts. Small white rectangles indicate regions magnified in bottom-right insets, showing colocalization of Z-RNA and FAM-labelled hairpins. c, Quantitation of colocalized Z-RNA and FAM in b. n = 11 fields (LINE, SINE), n = 10 fields (LTR, A-RNA and Z-RNA). d, Immunofluorescence staining of Z-RNA and FLAG in FLAG-ZBP1 MEFs transfected with RNA designed from aberrant Z-forming transcripts. Small white rectangles indicate regions that are magnified in bottom-right insets, showing colocalization of Z-RNA and FLAG-ZBP1. e, Quantitation of colocalized FLAG-ZBP1 and Z-RNA in c. n = 10 fields (LINE, LTR, A-RNA and Z-RNA), n = 8 fields (SINE). f, g, Kinetics of cell death in IFNβ pretreated (100 ng/mL, 16 h) primary Zbp1+/+ MEFs (f) or primary Zbp1–/– MEFs (g) transfected with the indicated RNA hairpins. h, Levels of CPSF3 protein in MEFs transfected with either control siRNA or CPSF3 siRNA. i, Z-RNA accrual in MEFs transfected with either control siRNA or CPSF3 siRNA. Cells were exposed to RNase A post-fixation, before staining with anti-Z-NA antibody. Nuclei are outlined with dashed white lines. j, Fluorescence intensity of Z-RNA signal in i. n = 35 cells (Si Ctrl), n = 34 cells (Si Cpsf3 and Si Cpsf3 + RNase A). k, Kinetics of cell death in Vec and FLAG-ZBP1 MEFs transfected with either control siRNA or CPSF3 siRNA. l, Alignment of JTE607-bound human CPSF3 crystal structure (bold) and murine CPSF3 AlphaFold model (transluscent). JTE-607 is shown as ball and stick (carbons; black, chlorine; green, oxygen; red, nitrogen; blue) with mesh (grey) representation to highlight occupied chemical space. The metallo-β-lactamase domain (green; residues 1-208 and blue; residues 396–459) and β-CASP domain (yellow; residues 209–395) display near identical folds, even in the absence of bound ligand. m, Active JTE-607 possesses the carboxylic acid moiety necessary for the bifurcated interaction with G330 and F241 backbone amides in CPSF3. Electrostatic interactions between the piperazine functional group of JTE-607 with E48 side chain and L47 backbone amide are also shown (orange dashes). n, Kinetics of cell death in IFNβ pretreated (100 ng/mL, 16 h) primary Zbp1+/+ and Zbp1–/– MEFs treated with DMSO (vehicle) or JTE-607 (100 μM), in the presence of zVAD (50 µM). Data are mean ± s.d. (n = 4 in f, g, k, n). One-way ANOVA with Dunnett’s multiple comparisons test (c, e, j). ***P < 0.0005 (P < 0.0001 in c, e, j). Data are representative of at least two independent experiments (b, d, h, i) or three independent experiments (f, g, k, n).
