Project description:CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported that CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA itself is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) which is required for the toxicity and processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi.
Project description: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.
Project description:Analysis of RISC bound short (s)RNAs in a HCT116 wild-type, Drosha k.o., and Dicer k.o. cells expressing pLenti-CD95L NP or pLenti empty vector.
Project description:Analysis of RISC bound short (s)RNAs in a HCT116 Drosha CD95 d.k.o. cell line expressing pLenti-CD95L and various CD95L mutant constructs.
Project description:Analysis of RISC bound short (s)RNAs in a HCT116 wild-type, Drosha k.o., and Ago 1/2/3 k.o. cells expressing pLenti-CD95L NP or pLenti empty vector.
Project description:Analysis of RISC bound short (s)RNAs in a HCT116 Drosha CD95 d.k.o. cell line expressing pLenti-CD95L and various CD95L mutant constructs.
