Non-coding YRNAs in Death Induced by Survival gene Elimination (DISE)

Death Induced by Survival gene Elimination (DISE) is a unique form of cell death that activates multiple cell death pathways in parallel. DISE occurs when toxic sequences embedded in the genes of the death receptor CD95 and its ligand CD95L induce a RNAi-mediated off-target effect that specifically targets the 3’ untranslated region (UTR) of survival genes. To identify other small RNA mediators of DISE, we isolated RNA <200 bp from two cell lines infected with shRNAs containing CD95/CD95L sequences in which we deleted the shRNA target site using CRISPR/Cas9 gene editing. An RNA-Seq analysis revealed that two highly conserved, small non-coding RNAs (ncRNAs), RNY4 and RNY5, were among the most highly upregulated RNA species in both cell lines. RNY4 and RNY5 belong to a family of RNAs called YRNAs. Full length YRNAs (RNY1, 3, 4 and 5) are thought to act as tethers and guides for various proteins, and have been implicated in several essential cell functions such as the initiating DNA replication, processing histone pre-mRNA 3’ UTRs, protecting embryonic stem cells from UV stress, and recognizing misfolded small nuclear RNA. Processed YRNA fragments are increased in the serum of cancer patients, and fragments of similar sequence were identified in our RNA-Seq analysis. We have identified specific RNY4 and RNY5 fragments that are upregulated during DISE induced by either CD95/CD95L derived shRNAs, by chemotherapeutic reagents and during CD95 mediated apoptosis. While full length YRNAs have been mapped to a cluster on chromosome (chr) 7 in humans, the fragments we detected are encoded in hundreds of locations on multiple chromosomes. Expression of YRNA fragments from multiple chromosomes has been reported during the lytic phase of EBV infection. We hypothesize that expression of these sequences from multiple chromosomes will be activated to contribute to DISE. We have generated CRISPR/Cas9 knockouts of chr7 encoded RNY4 and RNY5, and we will now determine the contribution of YRNAs from alternative chromosomes to DISE.