CD95/Fas ligand mRNA is toxic to cells through more than one mechanism

Abstract

CD95/Fas ligand (CD95L) induces apoptosis through protein binding to the CD95 receptor. However, CD95L mRNA also induces toxicity in the absence of CD95 through induction of DISE (Death Induced by Survival Gene Elimination), a form of cell death mediated by RNA interference (RNAi). We now report that CD95L mRNA processing generates a short (s)RNA nearly identical to shL3, a commercial CD95L-targeting shRNA that led to the discovery of DISE. Neither of the miRNA biogenesis proteins Drosha nor Dicer are required for this processing. Interestingly, CD95L toxicity depends on the core component of the RISC, Ago2, in some cell lines, but not in others. In the HCT116 colon cancer cell line, Ago 1–4 appear to function redundantly in RNAi. In fact, Ago 1/2/3 knockout cells retain sensitivity to CD95L mRNA toxicity. Toxicity was only blocked by mutation of all in-frame start codons in the CD95L ORF. Dying cells exhibited an enrichment of RISC bound (R)-sRNAs with toxic 6mer seed sequences, while expression of the non-toxic CD95L mutant enriched for loading of R-sRNAs with nontoxic 6mer seeds. However, CD95L is not the only source of these R-sRNAs. We find that CD95L mRNA may induce DISE directly and indirectly, and that alternate mechanisms may underlie CD95L mRNA processing and toxicity.

DISE, an ancient anti-cancer mechanism that senses mutational load in cancerous cells?

“…We subsequently reported that CD95L is enriched in sequences that when converted to sRNAs are toxic to cells. In fact, we found that CD95L is processed into short RNAs (sRNAs) that are loaded into the RNA induced silencing complex (RISC) and kill cells through DISE [ 10 , 11 ]. Later, we determined that all these toxic sRNAs did not kill cells by acting like typical siRNAs i.e., by silencing genes through complete complementarity to a section of the mRNA, but by working in a microRNA (miRNA)-like fashion i.e.…”