Figure 2. Stromal POL3-Derived ExoRNA Activates Breast Cancer RIG-I in a 5’-Triphosphate-Dependent Manner.
(A) Distribution of RNA classes found in cellular RNA and exosome RNA by RNA-seq after co-culture of1833 ISG-R breast cancer cells with MRC5 stromal cells. Ribosomal RNA counts were removed.
(B) ISG expression after transfection of co-culture exoRNA or co-culture cellular RNA into 1833 cells with wild-type RIG-I (WT), RIG-I knockout (KO), or RIG-I KO cells restored with either wild-type RIG-I (KO + WT) or RIG-IK858/861A (KO + MUT) (n = 5). Baseline was established by mock transfection (see legend).
(C) Immunoblot for POLR3G and ß-actin in sorted MRC5 fibroblasts after mono- or ISG-R co-culture (left) along with quantification of replicates (right).
(D and E) Expression of ISGs in sorted 1833cells(D) or(E) RT-mediated cell death in 1833 cells after co-culture with MRC5cells(CTL) or after siRNA knockdown of POLR3F in 1833 (BrCa), MRC5 (Strm), or both cell types (Both). Gene expression is relativeto1833 cells in mono-culture, and cell death was assessed4days after 10 Gy RT (n = 3).
(F) ISG expression in 1833 cells after addition of conditioned media (CM) from ISG-R co-cultures treated with DMSO or POL3 inhibitor (POL3i). Values are relative to 1833 cells in mono-culture (n = 3).
(G) RT-mediated cell death of 1833 cells in mono-culture (Mono) or co-culture with MRC5 cells (Co-cx). Cells were grown in the presence of DMSO or POL3i and with (+CM) or without CM from ISG-R co-culture (n = 3).
(H) ATP hydrolysis assay for recombinant RIG-I activation by increasing amount sof the indicated RNA. ExoRNA and cellular RNA are from 1833 ISG-R co-culture treated with or without POL3i (n = 3). 5’OH is a negative control and 5’ppp is a positive control.
(I) Abundance (Log10) of RNA classes from exoRNA sequencing compared to 5’ppp-seq. RNA classes depleted in 5’ppp-seq by ~10-fold or greater are shown on the left (n = 4).
(J) Relative levels of POL3 transcripts in exosomes harvested from ISG-R co-culture treated with DMSO or POL3i (n = 3). Error bars are SEM of biological replicates and *p < 0.05, **p < 0.01. See also Figure S2.