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

Bartom, Elizabeth T.; Kocherginsky, Masha; Paudel, Bidur; Vaidyanathan, Aparajitha; Haluck-Kangas, Ashley; Patel, Monal; O’Shea, Kaitlyn; Murmann, Andrea E.; Marynen, Peter

doi:10.1371/journal.pcbi.1010022

Cited by 10 publications

(24 citation statements)

References 36 publications

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“…R-sRNA were identified by an Ago1-4 peptide pulldown, and RNA precipitation combined with high-throughput sequencing (Ago-RP-Seq) as described [65]. Employing the SPOROS pipeline we developed to analyze RNA seq data in a seed based fashion [35] we found that the sRNAs that were significantly enriched in the RISC of cells treated with Aβ42 compared to cells treated with Aβ40 had a significantly lower seed viability than sRNAs that were depleted in the RISC ( Fig. 1E ).…”

Section: Resultsmentioning

confidence: 99%

“…Most analyses on AD associated miRNAs are based on quantifying miRNAs from total small RNA fraction and different miRNAs have been associated with the disease [90, 91]). We recently analyzed short RNAs in total RNA from a large cohort of AD patients [35]. Our data suggested that a substantial number of sRNAs other than miRNAs were upregulated in AD patient brains compared to controls.…”

Section: Discussionmentioning

confidence: 99%

“…With Ago2 expression levels unaffected the median seed toxicity across these R-sRNAs was much higher than R-sRNAs in wt cells [41]. The SPOROS bioinformatics pipeline was developed to visualized these changes in total and R-sRNAs and the effect of lack of Drosha expression on seed toxicity and cell fate was quantified by producing a number of graphical outputs [35]: 1) A seed toxicity graph that plots sRNAs according to their abundance (Y axis) and seed viability (X axis), based on the average seed viability on three human cell lines (see 6merdb.org), 2) The median seed viability of all sRNAs in form of a box plot, and 3) The average nucleotide composition of the 6mer seed. Thus, to investigate the effect of lack of Drosha on these brain derived cells we sequenced both the total cellular and the R-sRNAs from the parental cells and two Drosha k.o.…”

Section: A Neuroblastoma Cell Line With Reduced Levels Of Protective ...mentioning

confidence: 99%

“…More recently, we developed SPOROS, a bioinformatics tool to aid in the study of the role of this "kill code" in cell death more broadly. SPOROS is a standardized, semi-automated pipeline to analyze seed toxicity of sRNAs in a gene agnostic fashion [35].…”

Section: Introductionmentioning

confidence: 99%

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Death Induced by Survival gene Elimination (DISE) is correlated with neurotoxicity in Alzheimer’s disease and aging

Paudel

Jeong

Martinez

et al. 2022

Preprint

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Background: Alzheimer's 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 recently discovered powerful cell death mechanism mediated by short (s) RNAs including micro (mi) RNAs acting through the RNA induced silencing complex (RISC). G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich seed matches in genes essential for cell survival resulting in the simultaneous activation of multiple cell death pathways. The RISC of most cells is occupied by miRNAs with nontoxic 6mer seeds, which may protect them from DISE by blocking loading of toxic sRNAs. However, during aging when miRNA expression decreases, toxic sRNAs may enter the RISC more readily leaving cells primed for DISE. Whether DISE contributes to neuronal loss in a neurodegenerative disease such as AD has not been evaluated. Methods: Using Ago precipitation and RNAseq (Ago-RP-Seq) combined with SPOROS, a recently developed bioinformatics pipeline to analyze small RNAseq data with respect to 6mer seed toxicity, we analyzed RISC bound sRNAs (R-sRNAs) in in vitro models and in the brains of multiple in vivo AD mouse models, aged mice, and AD patients. Results: We find that in in vitro cell line studies, in mouse models that show neurodegeneration, and in the aging brain R-sRNAs shift to more toxic seeds. In contrast, in cells that survived in post-mortem brains of AD patients and the brains of "SuperAgers", individuals over age 80 who have superior memory performance, R-sRNAs shift to more nontoxic seeds, supporting a protective function of miRNAs. Conclusion: Our data provide first evidence of a contribution of DISE to the neurotoxicity seen in AD suggesting that increasing the levels of protective miRNAs in the brain or blocking the activity of toxic R-sRNAs could lead to a novel way of treating the disease.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: A Neuroblastoma Cell Line With Reduced Levels Of Protective ...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Death Induced by Survival gene Elimination (DISE) is correlated with neurotoxicity in Alzheimer’s disease and aging

Paudel

Jeong

Martinez

et al. 2022

Preprint

Self Cite

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“…Our recent development of the Ago-RP-Seq-SPOROS pipeline [30] allows us to explore the connection between the sRNAs in the RISC and cell death in a standardized way. By applying data from the 6mer seed viability screens, we have now studied the connection between RISC bound sRNAs (R-sRNAs), their predicted effect on cell viability through seed-based targeting, and the resultant phenotypic outcomes in more detail.…”

Section: Discussionmentioning

confidence: 99%

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

Haluck-Kangas

Fink

Bartom

et al. 2022

Preprint

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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 3′UTR 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 non-toxic 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.

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The length of uninterrupted CAG repeats in stem regions of repeat disease associated hairpins determines the amount of short CAG oligonucleotides that are toxic to cells through RNA interference

et al. 2022

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Extended CAG trinucleotide repeats (TNR) in the genes huntingtin (HTT) and androgen receptor (AR) are the cause of two progressive neurodegenerative disorders: Huntington’s disease (HD) and Spinal and Bulbar Muscular Atrophy (SBMA), respectively. Anyone who inherits the mutant gene in the complete penetrance range (>39 repeats for HD and 44 for SBMA) will develop the disease. An inverse correlation exists between the length of the CAG repeat and the severity and age of onset of the diseases. Growing evidence suggests that it is the length of uninterrupted CAG repeats in the mRNA rather than the length of poly glutamine (polyQ) in mutant (m)HTT protein that determines disease progression. One variant of mHTT (loss of inhibition; LOI) causes a 25 year earlier onset of HD when compared to a reference sequence, despite both coding for a protein that contains an identical number of glutamines. Short 21–22 nt CAG repeat (sCAGs)-containing RNAs can cause disease through RNA interference (RNAi). RNA hairpins (HPs) forming at the CAG TNRs are stabilized by adjacent CCG (in HD) or CUG repeats (in SBMA) making them better substrates for Dicer, the enzyme that processes CAG HPs into sCAGs. We now show that cells deficient in Dicer or unable to mediate RNAi are resistant to the toxicity of the HTT and AR derived HPs. Expression of a small HP that mimics the HD LOI variant is more stable and more toxic than a reference HP. We report that the LOI HP is processed by Dicer, loaded into the RISC more efficiently, and gives rise to a higher quantity of RISC-bound 22 nt sCAGs. Our data support the notion that RNAi contributes to the cell death seen in HD and SBMA and provide an explanation for the dramatically reduced onset of disease in HD patients that carry the LOI variant.

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

Cited by 10 publications

References 36 publications

Death Induced by Survival gene Elimination (DISE) is correlated with neurotoxicity in Alzheimer’s disease and aging

Death Induced by Survival gene Elimination (DISE) is correlated with neurotoxicity in Alzheimer’s disease and aging

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

The length of uninterrupted CAG repeats in stem regions of repeat disease associated hairpins determines the amount of short CAG oligonucleotides that are toxic to cells through RNA interference

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