Biology subjects

Peter, M. E.

Publications and source records attributed to Peter, M. E..

11 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

Contribution of 6mer seed toxicity to HIV-1 induced cytopathicity

HIV-1 (HIV) infects CD4 positive T cells, the gradual depletion of which can lead to the onset of Acquired Immunodeficiency Syndrome (AIDS) in the absence of antiretroviral therapy (ART). Several forms of cell death have been shown to be involved in HIV-mediated killing of either directly infected or bystander cells. It is still unknown, however, why some cells survive HIV infection and persist as part of the latently infected reservoir that reliably causes recurrent viremia upon ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could inform innovations designed to clear the latent reservoir. "Death Induced by Survival gene Elimination" (DISE) is an RNA interference (RNAi)-based mechanism that kills cells through short (s)RNAs with toxic 6mer seeds (pos. 2-7 of sRNA). These toxic seeds target reverse complementary seed matches in the 3UTR of mRNA transcripts to decrease expression of hundreds of genes that are critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded non-toxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. We now report that infection of cells with HIV results in RISC-loading of an HIV-encoded miRNA, v-miRNA HIV-miR-TAR-3p, which kills cells by DISE through a noncanonical (pos. 3-8) 6mer seed. In addition, cellular RISC bound sRNAs shift to lower seed viability. Both these effects also occur with latent HIV provirus reactivation in J-Lat cells, a well-established cell model of HIV latency. Cells lacking expression of miRNA biogenesis genes Drosha, Dicer and Exportin 5 cannot produce protective miRNAs and therefore do not block RISC loading of the v-miRNA HIV-miR-TAR-3p. These mutant cells, as well as cells lacking expression of the RISC component Ago2, are hypersensitive to cell death via DISE induced by HIV infection. More precise targeting of the balance between protective and cytotoxic sRNAs could specifically and transiently increase silencing of cell survival genes to increase DISE. This could be a new addition to a "shock and kill" strategy to enhance depletion of the provirus reservoir during suppressive ART.

microbiology↗

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↗

bioRxiv2021

CD95 expression in triple negative breast cancer blocks induction of an inflammatory state through differential regulation of NF-kB Signaling

CD95L is expressed by tumor-infiltrating lymphocytes to eliminate CD95-expressing tumor cells and thereby CD95 loss by tumor cells is often considered as a consequence of an immunoediting process. Nonetheless CD95 expression is maintained in most triple negative breast cancers (TNBCs), and we recently reported that CD95 loss in TNBC cells triggers the induction of a pro-inflammatory program promoting the recruitment of cytotoxic NK and CD8+ T-cells and impairing tumor growth. Using a comprehensive proteomic approach, we have identified two yet unknown CD95 interaction partners, Kip1 ubiquitination-promoting complex protein 2 (KPC2) and p65. KPC2 contributes to the partial degradation of p105 (NF{kappa}B1) and the subsequent generation of p50 homodimers, which transcriptionally represses pro-inflammatory NF-{kappa}B-driven gene expression. Mechanistically, KPC2 directly interacts with the C-terminal region of CD95 and links the receptor to RelA (p65) and KPC1, the catalytic subunit of the KPC complex that acts as E3 ubiquitin-protein ligase promoting the partial degradation of p105 into p50. Loss of CD95 in TNBC cells releases KPC2, limiting the formation of the NF-{kappa}B inhibitory homodimer complex (p50/p50), promoting NF-{kappa}B activation and the production of pro-inflammatory cytokines including CSF1, CSF2, CXCL1 and IL1 members, known to promote recruitment and differentiation of certain adaptive and innate immune effector cells.

cancer biology↗

bioRxiv2021

Tumor expressed CD95 causes suppression of anti-tumor activity of NK cells in a model of triple negative breast cancer

The apoptosis inducing receptor CD95/Fas has multiple tumorigenic activities. Stimulation by its cognate ligand CD95L on many cancer cells increases their growth, motility, ability to invade and/or their cancer stemness. Using genetically engineered mouse models of ovarian and liver cancer, we previously reported that deletion of CD95 in the tumor cells strongly reduced their ability to grow in vivo [1, 2]. Using a combination of immune-deficient and immune-competent mouse models, we now establish that loss of CD95 in metastatic triple negative breast cancer cells prevents tumor growth by modulating the immune landscape. CD95 deficient but not wild-type tumors barely grow in an immune-competent environment and show an increase in immune infiltrates into the tumor. This growth reduction is caused by NK cells and does not involve CD8+ T cells. On the other hand, in immune compromised mice CD95 k.o. cells are not growth inhibited, but they fail to form metastases. In summary, we demonstrate that in addition to its tumor and metastasis promoting activities, CD95 expression by tumor cells can exert immune suppressive activities providing a new target for immune therapy.

cancer biology↗

bioRxiv2021

6mer Seed Toxicity and Platinum Resistance in Ovarian Cancer

Numerous micro(mi)RNAs (short noncoding RNAs that negatively regulate gene expression) have been linked to platinum (Pt) sensitivity and resistance in ovarian cancer (OC). miRNA activity occurs when the guide strand of the miRNA, with its seed sequence (pos. 2-7/8), is loaded into the RNA induced silencing complex (RISC) and targets complementary short seed matches in the 3 untranslated region of mRNAs. Toxic seeds, targeting genes critical for cancer cell survival, have been found in tumor suppressive miRNAs. Many si- and shRNAs can also kill cancer cells via toxic seeds, the most toxic carrying G-rich 6mer seed sequences. We now show that treatment of OC cells with Pt leads to an increase in RISC-bound miRNAs carrying toxic 6mer seeds and a decrease in miRNAs with nontoxic seeds. Pt-resistant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in OC patients revealed that the ratio between miRNAs with toxic versus miRNAs with nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses.

cancer biology↗

bioRxiv2020

Identification of the toxic 6mer seed consensus in human cancer cells

6mer seed toxicity is a novel anti-cancer mechanism that kills cancer cells by triggering death induced by survival gene elimination (DISE). It is based on si- or shRNAs with a specific G-rich nucleotide composition in position 2-7 of their guide strand. An arrayed screen of 4096 6mer seeds on two human and two mouse cell lines identified a consensus GGGGGC as the most toxic seed. After testing two more cell lines, one human and one mouse, we found that the GGGGGC seed while also toxic to murine cells, is more toxic to human cells, suggesting that the evolution to use of Gs as part of the toxic seeds is still slowly evolving, with Gs more common in the human toxic seeds. While new RNA Seq and bioinformatics analyses suggest that the GGGGGC seed is toxic to cancer cells by targeting GCCCCC seed matches in the 3 UTR of a set of genes critical for cell survival, we now directly confirm this by identifying a number of genes targeted by this seed. Furthermore, by using a luciferase reporter fused to the 3 UTR of these genes we confirm direct and specific on-targeting of GCCCCC seed matches. Targeting is strongly attenuated after mutating the GCCCCC seed matches in these 3 UTRs. Our data confirm that an siRNA containing the GGGGGC seed kills cancer cells through its miRNA like activity and points at artificial miRNAs, si- or shRNAs containing this seed as a potential new cancer therapeutics.

cancer biology↗

bioRxiv2019

6mer seed toxicity in viral microRNAs

Micro(mi)RNAs are short double stranded noncoding RNAs (19-23nts) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just 6 nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposis sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.

cell biology↗

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↗

bioRxiv2018

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntingtons disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complimentary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the 6 members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anti-cancer reagents.

cancer biology↗