Biology subjects

Haluck-Kangas, A.

Publications and source records attributed to Haluck-Kangas, A..

6 recordsLinked to original sources

bioRxiv2022

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

CD95/Fas ligand induces apoptosis through binding of the protein to the CD95 receptor. However, CD95L mRNA also induces toxicity in the absence of CD95. Dying cells exhibit features of DISE (Death Induced by Survival Gene Elimination), a form of cell death mediated by RNA interference (RNAi). DISE relies on targeting mediated by six nucleotides of complementarity between positions 2-7, the 6mer seed sequence of a RISC-bound (R-sRNA), and the 3UTR of an mRNA, a feature that allows to predict the effect of 6mer seed sequences on cell viability. We now report that CD95L mRNA processing generates an sRNA nearly identical to shL3, a commercial CD95L-targeting shRNA that led to the discovery of DISE. Neither of the miRNA biogenesis proteins Drosha or Dicer are required for CD95L mRNA processing. Interestingly, CD95L toxicity depends on the core component of the RISC, Ago 2, 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 retained sensitivity to CD95L mRNA toxicity. Toxicity was only blocked by mutation of all in-frame start codons in the CD95L ORF. Expression of a toxic CD95L mRNA caused an enrichment for R-sRNAs with toxic 6mer seed sequences, while expression of the nontoxic CD95L mutant enriched for loading of R-sRNAs with nontoxic 6mer seeds. However, CD95L was not the only source of these R-sRNAs. We found that CD95L mRNA may induce DISE directly and indirectly, and that alternate mechanisms may underlie CD95L mRNA processing and toxicity.

cell biology↗

bioRxiv2022

Death induced by survival gene elimination (DISE) contributes to neurotoxicity in Alzheimer's disease

Alzheimers disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to A{beta}42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of "SuperAgers", humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to A{beta}42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and A{beta}42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration.

neuroscience↗

bioRxiv2021

SPOROS: A pipeline to analyze DISE/6mer seed toxicity

micro(mi)RNAs are (18-22nt long) noncoding short (s)RNAs that suppress gene expression by targeting the 3 untranslated region of target mRNAs. This occurs through the seed sequence located in position 2-7/8 of the miRNA guide strand, once it is loaded into the RNA induced silencing complex (RISC). G-rich 6mer seed sequences can kill cells by targeting C-rich 6mer seed matches located in genes that are critical for cell survival. This results in induction of Death Induced by Survival gene Elimination (DISE), also referred to as 6mer seed toxicity. miRNAs are often quantified in cells by aligning the reads from small (sm)RNA sequencing to the genome. However, the analysis of any smRNA Seq data set for 6mer seed toxicity requires an advanced workflow, solely based on the exact position 2-7 of any sRNA that can enter the RISC. Therefore, we developed SPOROS, an automated pipeline that produces multiple useful outputs to compare 6mer seed toxicity of all cellular sRNAs, regardless of their nature, between different samples. We provide two examples to illustrate the capabilities of SPOROS: Example one involves the analysis of RISC-bound sRNAs in a cancer cell line (either wild-type or two mutant lines unable to produce most miRNAs). Example two is based on a publicly available smRNA Seq data set from postmortem brains (either from normal or Alzheimers patients). Our methods are designed to be used to analyze a variety of smRNA Seq data in various normal and disease settings.

bioinformatics↗

bioRxiv2018

CD95L mRNA is toxic to cells

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported the 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) RISC which is required for the toxicity and that 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.

cancer biology↗

bioRxiv2017

Induction of DISE in ovarian cancer cells in vivo

The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nano particles (TLP). In vitro, TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo, two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

cancer biology↗

bioRxiv2017

CD95L mRNA And CD95L Derived si- and shRNAs Kill Cancer Cells Through An RNAi Mechanism By Targeting Survival Genes

Over 80% of multiple tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that using specific toxic RNAi-active sequences present in the genome can kill cancer cells.

cancer biology↗