Figure 4. R-loop-derived cytoplasmic RNA-DNA hybrids trigger IRF3 signaling via the cGAS and TLR3 receptors.

(a) Dependence of SETX knockdown-induced pIRF3 level on cGAS or TLR3 in HeLa cells.

(b) RT-qPCR measurements of IRF3 effectors upon knockdown of SETX and TLR3 and cGASi treatment in HeLa cells.

(c) Western blots of C-PARP upon perturbation of immune receptors in SETX- or BRCA1-depleted HeLa cells.

(d) Western blot of pIRF3 upon TLR3 or cGAS loss after synthetic RNA:DNA hybrid transfection.

(e) Gel shift assay showing in vitro binding of cGAS and TLR3 to RNA-DNA hybrids. NP: no protein.

(f) In vitro pull-down assay showing hybrid binding activity of purified cGAS and TLR3. dRH and GFP are positive and negative controls respectively.

(g) Coimmunoprecipitation of cGAS or TLR3 with cytoplasmic hybrids upon knockdown of SETX or BRCA1, with or without XPG knockdown.

(h) cytoDRIP blot of TLR3-associated hybrids in the cytoplasm of control and SETX-depleted HeLa cells, with mock and RH treatment prior to hybrid pull-down. dsDNA markers are indicated in bp.

(i) LysoIP blot shows RNA-DNA hybrid levels in the endolysosome of control and SETX-depleted HA-TMEM192 HEK293T cells, with or without RH treatment prior to hybrid pull-down. Flag-TMEM192 HEK293T cells: mock control.

(j) RT-qPCR measurements of IFNβ and ISGs in CTRL and AOA2 fibroblasts upon knockdown of XPG.

(k) As in (j) upon cGAS inhibition and TLR3 knockdown.

(l) cytoDRIP blot showing cytoplasmic hybrid production upon XPG knockdown in UWB1.289 and UWB1.289+BRCA1 cells.

(m) Western blot of pIRF3 and C-PARP in UWB1.289 and UWB1.289+BRCA1 cells stably expressing GFP (mock) and RH-NES.

Bar graphs represent the mean ± s.d. n = 3 independent biological replicates (two-tailed t-test with CI = 95%). P values are shown at the top of the graphs.