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.
| Original language | English (US) |
|---|---|
| Article number | 11 |
| Journal | Molecular Biomedicine |
| Volume | 4 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2023 |
Funding
We grateful for help from Sergii Pshenychnyi, Ph.D. and the Northwestern University—Recombinant Protein Production Core for help with optimizing production of the GST-T6B-flag peptide and to Monal Patel, Ph.D. for help with qPCR analyses. We also would like to thank Denise Scholtens, Ph.D. and Charles Lang, Ph.D. for biostatistics support, and members of the Peter lab for critical discussions of results. This work was supported by the National Institutes of Health (R35CA197450) and (T32CA009560). This work was also supported by the Northwestern University – Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). Flow Cytometry Cell Sorting was performed on a BD FACSAria SORP system and BD FACSymphony S6 SORP system, purchased through the support of NIH 1S10OD011996-01 and 1S10OD026814-01. Research reported in this publication was also supported by Northwestern University Skin Biology & Diseases Resource-Based Center of the National Institutes of Health under award number P30AR075049.
Keywords
- Cell death
- FasL
- RISC
- RNA toxicity
- RNAi
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology
