Extended Data Fig. 4: Analysis of RNA recognition by insect cGLRs.
From: cGAS-like receptors sense RNA and control 3′2′-cGAMP signalling in Drosophila
a–c, In vitro activity assays for each active insect cGLR demonstrating that dsRNA recognition is required for enzyme activation. Reactions were performed with 40-nt or 40-bp synthetic ligands. Weak Deu-cGLR ssRNA-stimulated activity may be explained by transient short duplex formation similar to observations that some ssDNA oligos can stimulate mouse cGAS dsDNA-dependent activity3. b, TLC and quantification for enzyme activation in the presence of a panel of 10–40-bp synthetic dsRNA ligands. dsRNA (30 bp) is sufficient to stimulate maximal activity for Tc-cGLRs, Dm-cGLRs and Lc-cGLRs, while Ds-cGLR1 requires 35 bp and Deu-cGLR can be activated by dsRNAs as short as 15 bp. Data are mean ± s.e.m., quantified relative to maximum observed activity. c, Reactions with 146-bp in vitro-transcribed dsRNAs containing either a 5′ triphosphate or 5′ OH termini demonstrate that dsRNA recognition by insect cGLRs does not involve 5′-end discrimination. d, Deconvolution of catalytic metal requirements for enzymatic activity by insect cGLRs. Insect cGLRs require Mn2+ for maximal catalytic activity, with weak product formation observed in the presence of Mg2+. e, Poly I:C titration demonstrates that dsRNA stimulation of Drosophila cGLR1 activity in cells is dependent on RNA concentration. IFNβ luciferase assay in which cGLRs are expressed in human cells and CDN synthesis is measured by mammalian STING activation, as in Fig. 2e and Extended Data Fig. 3f. IFNβ quantified relative to the empty vector control. Data are mean ± s.e.m. of n = 3 technical replicates. All data in a–e represent n = 3 independent experiments.
