CD95/Fas ligand mRNA is toxic to cells through multiple mechanisms

Haluck-Kangas, Ashley

doi:https://doi.org/10.21985/n2-ftrj-nx71

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CD95/Fas ligand mRNA is toxic to cells through multiple mechanisms

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CD95/Fas ligand (CD95L) is a well characterized activator of extrinsic apoptosis. CD95L protein binds to its cognate receptor, CD95/Fas/APO-1, inducing apoptotic signaling in sensitive cells. However, expression of CD95L is toxic even in the absence of CD95. We previously reported that the CD95L open reading frame (ORF) is enriched for sequences that when converted to short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) kill cells through Death Induced by Survival gene Elimination (DISE). DISE results from the preferential targeting of survival genes by short RNA (sRNA) with guanine-rich 6mer seed sequences (positions 2-7) loaded into the RNA-induced silencing complex (RISC). RISC-bound sRNA (R-sRNA) target cytosine-rich regions in the 3’ untranslated region (UTR) of survival genes inducing a biochemically and morphologically distinct form of cell death, DISE. Expression of CD95L mRNA in CD95 knockout cell lines induced cell death that resembled DISE. CD95L mRNA was processed into R-sRNAs and one was found to be identical in sequence to, shL3, an shRNA that led to the discovery of DISE. However, catalytic components of the miRNA pathway, Dicer and Drosha are not required for CD95L processing. In fact, their absence sensitized cells to DISE. HCT116 Drosha and Dicer knock-out cells, nearly devoid of miRNAs, exhibited increased loading of R-sRNAs derived from CD95L and from hundreds of other mRNAs, with preferential loading of sRNA derived from genes involved in translation. Knockdown of Ago2 rescued CD95L mRNA toxicity in an ovarian and a breast cancer cell line, however we found that the role of Ago2 in mediating CD95L mRNA toxicity may be cell type specific, as neither knockdown nor knockout of Ago2 rescued CD95L mRNA toxicity in the colon cancer cell line HCT116. Knockout of Ago2 also did not block shL3 induced toxicity in this cell line suggesting that other Ago genes may functionally compensate for the loss of Ago2. In fact, changes in the 6mer seed viability of Ago bound sRNAs support a role for RNAi in CD95L mRNA toxicity. Shifts in the 6mer seed viability of R-sRNAs correlated with the functional effects of CD95L mRNA expression. Interestingly, while mutating 303 synonymous nucleotide positions in CD95L did not eliminate toxicity, mutating just 13 in-frame ATG codons rendered CD95L mRNA nontoxic. Expression of this nontoxic CD95L mutant also did not induce a toxic shift in R-sRNAs. This may suggest a role for the translational machinery in mediating the toxicity of CD95L mRNA. We hypothesize that CD95L mRNA may exert toxicity on cells through various mechanisms including the loading of toxic sRNAs into the RISC leading to the activation of DISE.

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