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. 2024 Jul 9;57(7):1482–1496.e8. doi: 10.1016/j.immuni.2024.04.010

Figure 3.

Figure 3

RNase T2 and PLD enzymes release 2′,3′-cyclic GMP

(A and B) Fluorescence intensity signal of FAM-RNA40-BMN-Q530 over time, incubated with indicated concentrations of (A) PLD3 or (B) PLD4. Data are depicted as mean of n = 3 independent experiments.

(C) Overlay of extracted ion chromatograms (EIC) of 5′-GMP, 3′-GMP, and 2′,3′-cyclic GMP. Top: standards used as reference. Middle: in vitro digests of RNA9.2s with PLD3 (250 nM). Bottom: PLD4 (250 nM).

(D) Normalized signal areas of EIC from released 5′-NMPs, 3′-NMPs, and 2′,3′-cNMPs by PLD3 (250 nM) and PLD4 (250 nM) after degradation of RNA9.2sO in %.

(E and F) CAL-1 WT cells (300,000 cells/well) were stimulated with increasing concentrations of R848 and 2′,3′-cGMP. After 16 h, IFN-β release was determined by ELISA. Each replicate of n = 2 (E) or n = 3 (F) independent experiments is depicted. A four-parameter dose-response curve was fitted to calculate half-maximal effective concentration (EC50).

(G) CAL-1 cells (300,000 cells/well) of indicated genotypes were unstimulated or stimulated with indicated concentrations of 2′,3′-cGMP and 3′-GMP. Data are depicted as mean ± SEM of three independent experiments.

(H) Schematic view of the in vitro digestion assay.

(I) RNA40O was digested with RNase T2 (370 nM), PLD3 (250 nM), and PLD4 (250 nM) or in combinations of RNase T2 (370 nM) with either PLD3 (250 nM) or PLD4 (250 nM) for 20 min, and the release of 2′,3′-cGMP was analyzed by LC-MS.

(J and K) RNA40O was digested with RNase T2 (370 nM) in combination with either PLD3 (+ = 2.5 nM), PLD4 (+ = 50 nM), or with PLD4 (++ = 195 nM) for 20 min, and the release of 2′,3′-cGMP was analyzed by LC-MS.

(L) RNA40O was digested with RNase 1 (5.7 nM), RNase 2 (27 nM), RNase 6 (29 nM), or RNase T2 (37 nM) only or in combination with PLD3 (25 nM) for 30 min, and the release of 2′,3′-cGMP was analyzed by LC-MS. For (I), (J), (K), and (L), data are depicted as mean ± SEM of n = 3 independent experiments.

(M) Detection of 2′,3′-cGMP in cell lysates of RNA40O-stimulated WT, RNase T2, or PLD3−/− × PLD4−/− CAL-1 cells by LC-HRMS. Data are depicted as mean ± SEM of n = 3 independent experiments. Statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparison tests.