1. Cancer Biology
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CD95/Fas ligand mRNA is toxic to cells

  1. Will Putzbach
  2. Ashley Haluck-Kangas
  3. Quan Q Gao
  4. Aishe A Sarshad
  5. Elizabeth T Bartom
  6. Austin Stults
  7. Abdul S Qadir
  8. Markus Hafner
  9. Marcus E Peter  Is a corresponding author
  1. Feinberg School of Medicine, Northwestern University, United States
  2. National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, United States
  3. Northwestern University, United States
Research Advance
Cite this article as: eLife 2018;7:e38621 doi: 10.7554/eLife.38621
5 figures, 1 table, 2 data sets and 2 additional files

Figures

Figure 1 with 3 supplements
The CD95L mRNA is toxic to cells.

(A) Left: Schematic of the different CD95L mutants used. Right: Percent cell confluence over time of HeyA8 parental cells in the absence (left panel) or in the presence of 20 μM zVAD-fmk (center panel), or CD95 k.o. cells (right panel) after expression of CD95L constructs. Data are representative of one to three independent experiments. Values were calculated from samples done in triplicate or quadruplicate shown as mean ±SE. (B) Left: Western blot analysis of HeyA8 cells overexpressing different CD95L mutant RNAs. Cells expressing CD95LMUT or CD95L were pretreated with 20 μM zVAD-fmk. Note the small amount of truncated CD95L in cells infected with CD95L MUTNP does not have CD95 binding activity. Very similar data were obtained when the constructs were expressed in either CD95 k.o. HeyA8 cells (clone #11) or NB-7 cells, which lack expression of caspase-8, both without treatment with zVAD (data not shown). Right: RT-qPCR analysis for CD95L of the same samples. Data are representative of two independent experiments. Each bar represents mean ±S.D. of three replicates. (C, D) Quantification of cell death (C) and ROS production (D) in CD95 k.o. HeyA8 cells (clone #11) expressing either pLenti (v) or pLenti-CD95L (L) at different time points (days after infection). Data are representative of two independent experiments. Each bar represents mean ±SE of three replicates. *p<0.05, **p<0.001, ***p<0.0001, unpaired t-test. (E) Confluency over time of the MCF-7 complete CD95 k.o. FA4 clone (right) or a MCF-7 clone #21 in which we deleted the shR6 target site resulting in an out-of-frame shift after infection with either pLenti vector control (vec) or wt CD95L. Data are representative of two independent experiments. Each data point represents mean ±SE of three replicates.

https://doi.org/10.7554/eLife.38621.002
Figure 1—figure supplement 1
Generation of complete CD95 k.o. MCF-7 cells.

(A) Schematic of the genomic locations and sequences of the gRNAs used to excise the entire CD95 gene in MCF-7 cells. PAM sites are underlined. (B) PCR with flanking (top panels) and internal (bottom panels) primers used to confirm the absence of the CD95 gene in MCF-7 clones. Parental (Par.) cells and three clones infected with Cas9 only (Cas9) and two homozygous complete CD95 k.o. clones (F2 and FA4) are shown. (C) RT-qPCR analysis of the indicated clones using primers spanning either exon 1/2 or exon 2/3 of the CD95 gene. (D) Surface staining for CD95 of one wt and one k.o. clone. (E) Western blot analysis of all clones.

https://doi.org/10.7554/eLife.38621.003
Figure 1—figure supplement 2
Mutation of the alternative start codon in CD95LMUTNP construct.

(A) Left: Schematic of the different CD95L mutants used. For the M86I mutant protein the point mutation on the DNA level is in parentheses. (B) Western blot analysis of HeyA8 CD95 k.o. cells overexpressing different CD95L mutants. Note, for unknown reasons in this experiment expression of the CD95LMUTNP protein was more efficient than in the experiment shown in Figure 1B. (C) Percent cell confluence over time of HeyA8 CD95 k.o. (cl. #11) cells after expression of the different CD95L constructs. Values were calculated from samples plated in triplicate shown as mean ±SE.

https://doi.org/10.7554/eLife.38621.004
Figure 1—figure supplement 3
Toxicity of CD95L mRNA is independent of CD95L protein expression.

(A) Schematic showing the positions of the codon optimized silent mutations of the CD95LSIL mutant compared to wild type CD95L (mutations highlighted in blue). (B) Percent cell confluence over time of HeyA8 CD95 k.o. cells (cl. #11) over-expressing empty pLenti vector (vec), wild-type CD95L (from two separately cloned viruses), or the CD95LSIL. (C) RT-qPCR analysis and Western blot (inset) of wild type CD95L and CD95LSIL mutant mRNAs in the over-expressing cells shown in B. (D) Probability density plot comparing the toxicity of all possible 6mer seeds located in either the WT or SIL CD95L mRNA. p-value was calculated using a two-sample two-sided K-S test.

https://doi.org/10.7554/eLife.38621.005
Figure 2 with 4 supplements
Toxicity induced by CD95L overexpression is reminiscent of DISE.

(A) Phase-contrast images of HeyA8 and HeyA8 CD95 k.o. cells (cl. #11) after infection with pLKO-shScr/shL3 or pLenti (vec)/pLenti-CD95L, respectively, at the indicated time point. (B) Gene set enrichment analysis for the 1846 survival genes (top panel) and the 416 nonsurvival genes (bottom panel) identified in the Sabatini study (Putzbach et al., 2017; Wang et al., 2015) of mRNAs downregulated in CD95L expressing HeyA8 CD95 k.o. cells compared to HeyA8 CD95 k.o. cells infected with pLenti virus. p-values indicate the significance of enrichment. (C) Common genes downregulated in all RNA-Seq experiments from (HeyA8) cells treated with either one of four si/shRNAs (Putzbach et al., 2017) derived from either CD95 or CD95L (see Figure 2D) and cells overexpressing CD95L ORF as described in (B). (D) Metascape analysis of 5 RNA Seq data sets analyzed. The boxed GO term clusters were highly enriched in all five data sets.

https://doi.org/10.7554/eLife.38621.006
Figure 2—video 1
CD95 k.o. HeyA8 cells (cl. #11) infected with pLenti control virus.
https://doi.org/10.7554/eLife.38621.007
Figure 2—video 2
CD95 k.o. HeyA8 cells (cl. #11) infected with pLenti-CD95L virus.
https://doi.org/10.7554/eLife.38621.008
Figure 2—video 3
HeyA8 cells infected with pLKO-shScr.
https://doi.org/10.7554/eLife.38621.009
Figure 2—video 4
HeyA8 cells infected with pLKO-shL3.
https://doi.org/10.7554/eLife.38621.010
Figure 3 with 1 supplement
Small RNAs generated in cells expressing CD95L mRNA are loaded into the RISC.

(A) Percent cell confluence over time of HCT116 parental (left) or Drosha k.o. (right) cells after infection with CD95MUTNP. Data are representative of three independent experiments. Each data point represents the mean ±SE of three replicates. Inset: Phase contrast images of Drosha k.o. cells 9 days after infection with either empty vector or CD95LMUTNP. (B) Percent cell confluence of HeyA8 CD95 k.o. cells transfected with either non-targeting siRNA (siCtr) or a pool of 4 siRNAs targeting AGO2 following subsequent infection with either empty pLenti (vec) or pLenti CD95L. Inset: Western blot showing knock-down of human AGO2. (C) Top: autoradiograph on RNAs pulled down with the Ago binding peptide. Bottom: Western blot analysis of pulled down Ago proteins. v, pLenti; L, pLenti-CD95L expressing cells. (D) Pie charts showing the relative ratio of sRNAs pulled down with the Ago proteins in wt and Drosha k.o. cells. Depicted are all the amounts of all sRNAs that contributed at least 0.01% to the total RNA content. Only in the Drosha k.o. cells was a significant amount of CD95L derived Ago bound reads found. They represented the 75th most abundant sRNA species (arrow). The average number of total sequenced reads (of two duplicates) are shown for each condition. (E) Top: Number of reads (normalized per million) of the top six most abundant sRNAs in the RISC of either HCT116 wt-pLenti or -pLenti-CD95L cells. Bottom: Number of reads (per million) of the top five genes with sRNAs most abundant in the RISC or of CD95L in the RISC of either HCT116 Drosha k.o. pLenti (v), or -pLenti-CD95L (L) cells. Note: miR-21 is not included as it is already shown in the top row. Bottom right panel: Abundance of Ago bound CD95L derived sRNAs. Shown in all panels is the abundance of RNAs in the four samples. Rep 1 and Rep 2, replicate 1 and 2.

https://doi.org/10.7554/eLife.38621.011
Figure 3—figure supplement 1
All CD95L mRNA mutants are toxic through RNAi.

(A) Percent cell confluence of HeyA8 CD95 k.o. cells (cl. #11) transfected with either non-targeting siRNAs (siCtr) or a pool of 4 siRNAs targeting AGO2 following subsequent infection with either empty pLenti (vec), pLenti CD95LMUTNP (left), or pLenti CD95LSIL (right). (B) Percent cell confluence of parental HeyA8 cells transfected with either a pool of 4 non-targeting siRNA (siCtr) or a pool of 4 siRNAs targeting AGO2 following subsequent infection with either empty pLenti (vec), pLenti CD95L WT (left), or pLenti CD95LSIL (right). (C) Percent cell confluence of parental HeyA8 cells transfected with either a pool of 4 non-targeting siRNA (siCtr) or a pool of 4 siRNAs targeting AGO2 following subsequent infection with either empty pLenti (vec), or pLenti CD95LMUTNP. In A cells were plated after puromycin selection. In (B) and (C) cells were plated directly after viral infection and puromycin was added 50 hr after infection (arrow).

https://doi.org/10.7554/eLife.38621.012
Figure 4 with 2 supplements
The entire CD95L mRNA gives rise to sRNAs that bind to the RISC.

(A) Length distribution of CD95L derived reads in various analyses. (B, C) Read alignment with CD95LMUTNP ORF of analyses of sRNAs pulled down with Ago proteins from HCT116 wt (B, top) and Drosha k.o. (B, bottom) cells and of total sRNAs from HCT116 Drosha k.o. cells (C) after infection with CD95LMUTNP. (D, E) Read alignment with wt CD95L ORF of analyses of sRNAs pulled down with Ago proteins (D) or total sRNAs (E) from HeyA8 CD95 k.o. cells after infection with wt CD95L. (F) Percent cell confluence over time of HCT116 parental (top) or Dicer k.o. (clone #43) (bottom) cells after infection with CD95MUTNP. (Dicer k.o. clone #45, gave a similar result, data not shown). Data are representative of two independent experiments. Each data point represents the mean ±SE of three replicates. (G) RT-qPCR analysis of clusters 8 and 21 in HCT116 parental, Dicer k.o. (clone #43), and Drosha k.o. cells after infection with CD95MUTNP. Each bar represents mean ± S.D. of three replicates. v, vector, L, CD95L expressing cells.

https://doi.org/10.7554/eLife.38621.013
Figure 4—figure supplement 1
Predicted secondary structure of CD95L ORF and toxicity of CD95L-derived sRNAs after conversion to siRNAs.

(A) The CD95LMUTNP RNA was subjected to a RNA secondary structure analysis (http://rna.tbi.univie.ac.at) using default settings. The locations of 22 reads representative of the 22 read clusters are shown. Regions with potential duplex formation are boxed. The oligonucleotides that were found to be toxic when expressed as siRNAs are circled. (B) Toxicity of the eight siRNAs designed using the CD95L-derived small RNA fragments bound to Ago as the antisense strand sequences 96 hr post-transfection in the indicated cell lines. Each data point represents the mean ±SE of three replicates.

https://doi.org/10.7554/eLife.38621.014
Figure 4—figure supplement 2
CD95L fragments are less toxic than full length CD95L mRNA.

(A) Schematic of the different CD95L fragments used. (B) Left: Western blot analysis of HeyA8 CD95 k.o. cells overexpressing different CD95L mutant RNAs. Right: RT-qPCR analysis for CD95L of the same samples using primers detecting either the 5' or the 3' half of the mRNA. Data are representative of two independent experiments. Each bar represents mean ±S.D. of three replicates. (C) Percent cell confluence over time of HeyA8 CD95 k.o. cells after expressing the CD95L mutant or fragments. Data are representative of two independent experiments. Values were calculated from samples done in triplicate shown as mean ±SE. (D) Left: Western blot analysis of HCT116 Drosha k.o. cells overexpressing different CD95L mutant RNAs. Right: RT-qPCR analysis for CD95L of the same samples using primers detecting either the 5' or the 3' half of the mRNA. Data are representative of two independent experiments. Each bar represents mean ±S.D. of three replicates. (E) Percent cell confluence over time of HCT116 Drosha k.o. cells after expressing the CD95L mutant or fragments. Data are representative of two independent experiments. Values were calculated from samples done in triplicate shown as mean ±SE.

https://doi.org/10.7554/eLife.38621.015
Figure 5 with 3 supplements
Endogenous mRNAs are processed and loaded into the RISC.

(A) Length distribution of reads derived from five of the top ten most abundant genes loaded into the RISC of CD95L expressing HCT116 Drosha k.o. cells. The numbers in parentheses indicate the ranking in the top ten most abundant genes with Ago bound reads. (B) Alignment of the reads from the five genes shown in A with horizontal blue lines representing the mapped positions of the reads. Each blue line represents an individual read, with its length in the plot proportional to the read length. Small RNAs pulled down with Ago proteins (top) or total sRNAs (bottom) from HCT116 and Drosha k.o. cells after infection with wt CD95L. *This stack contains 14899 reads of which 3000 were randomly chosen and plotted. (C) All 4262 genes in HCT116 Drosha k.o. cells expressing CD95L ranked according to highest expression with more than 10 reads expressed as reads per kb per million (RPKM). The abundance of the six genes shown in A and B is labeled. (D) Genes ranked according to highest abundance in the RISC of Drosha k.o. cells. Reads derived from the five genes in A are labeled as well as the location of the reads derived from CD95L. (E) Percent cell confluence over time of parental HCT116 and Drosha k.o. cells. (F) Average seed toxicity of all Ago-bound miRNAs and non-miRNAs (Other) in parental HCT116 and Drosha k.o. cells. Reads are shown as reads per million (RPM). Chi squared test was used to calculate p-value. (G) Percent cell confluence over time of Drosha k.o. HCT116 cells 24 hr after transfection with 25 nM of either nontargeting SMARTpool (siCtr) or AGO2 SMARTpool siRNAs. Each data point represents mean ±SE of three replicates. The experiment is representative of three biological repeats.

https://doi.org/10.7554/eLife.38621.016
Figure 5—figure supplement 1
Endogenous mRNAs are processed and loaded into the RISC - additional genes.

(A) Length distribution of reads derived from 5 of the top ten most abundant genes loaded into RISC of CD95L expressing HCT116 Drosha k.o. cells. The numbers in brackets indicate the ranking in the top ten most abundant genes with Ago bound reads. (B) Alignment of the reads from the five genes shown in A with their respective mRNAs derived from small RNAs pulled down with Ago proteins (top) or total small RNAs (bottom) from HCT116 and Drosha k.o. cells after infection with WT CD95L.

https://doi.org/10.7554/eLife.38621.017
Figure 5—figure supplement 2
Mapping of Ago bound reads from five processed genes to the human genome.

Alignment of all reads derived from the five genes shown in Figure 5A and B with the human genome. Shown are all 8 tracks of HCT116 wt and HCT116 Drosha k.o. cells infected with either pLenti control vector (vec) or pLenti-CD95L in duplicate. For each of the genes the Y axis was fixed to the same scale.

https://doi.org/10.7554/eLife.38621.018
Figure 5—figure supplement 3
Genes with multiple reads bound to Ago proteins are involved in cell growth and protein translation.

(A) All 558 protein coding genes that were processed similar to CD95L and had reads bound to Ago proteins were subjected to a DAVID GO analysis. Shown are all significantly enriched gene clusters. The top two clusters (dark green) stood out with very low p-values of enrichment. Clusters highlighted in green are connected to cell proliferation (cell cycle, anti-apoptosis or protein translation). (B) All 5629 genes with Ago bound reads (10 or more counts) ranked according highest abundance. The positions of the mRNAs of ribosomal proteins are indicated in red in the ranked mRNAs.

https://doi.org/10.7554/eLife.38621.019

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)CD95LNANM_000639
Gene (H. sapiens)CD95NANM_000043
Cell line (H. sapiens)MCF-7ATCCATCC: HTB-22Human adenocarcinoma of the mammary
gland, breast; derived from metastatic site:
pleural effusion
Cell line (H. sapiens)MCF-7 CD95
ΔshR6 clone
#21
this paperNAMCF-7 CD95 ΔshR6 clone #21 with homozygous
227 nucleotide deletion of the shR6 target
site in CD95 (chr10:89,008,920–89,009,146;
Human Dec. 2013 GRCh38/hg38 assembly)
produced using CRISPR/Cas9 technology;
verified homozygous CD95 protein knockout
Cell line (H. sapiens)MCF-7 CD95
deletion
clone FA4
this paperNAMCF-7 CD95 deletion clone FA4 with a
homozygous deletion of the entire
CD95 gene (chr10:88,990,657–89,015,785;
Human Dec. 2013 GRCh38/hg38 assembly)
produced using CRISPR/Cas9 technology;
verified homozygous CD95 protein knockout
Cell line (H. sapiens)HeyA8PMID: 4016745RRID: CVCL_8878Human high grade ovarian serous
adenocarcinoma; derived from parent
Hey cells (RRID: CVCL_0297)
Cell line (H. sapiens)HeyA8 shR6 k.o.
clone #11, HeyA8
CD95 k.o.
PMID: 29063830NAHeyA8 CD95 k.o. clone with a homozygous
227 nucleotide deletion of the shR6 target
site in CD95 (chr10:89,008,920–89,009,146;
Human Dec. 2013 GRCh38/hg38 assembly)
produced using CRISPR/Cas9 technology;
verified homozygous CD95 protein knockout
Cell line (H. sapiens)HCT116Korean Collection
for Type Cultures
(KCTC)
KCTC: cat#HC19023;
ATCC: CCL_247
Human colorectal carcinoma
Cell line (H. sapiens)Drosha-/-;
Drosha-/-
clone #40
Korean Collection for
Type Cultures (KCTC);
PMID: 26976605
KCTC: cat#HC19020HCT116 clone #40 with homozygous
knockout of Drosha protein; knockout
achieved using CRISPR/Cas9 which resulted
in a single nucleotide insertion in one allele and
a 26 nucleotide deletion in the other
Cell line (H. sapiens)Dicer-/-;
Dicer-/-
clone #43
Korean Collection for
Type Cultures (KCTC);
PMID: 26976606
KCTC: cat#HC19023HCT116 clone #43 with homozygous
knockout of Dicer protein; knockout
achieved using CRISPR/Cas9 which resulted
in a three nucleotide insertion and 14
nucleotide deletion in one allele and a
35 nucleotide deletion in the other
Cell line (H. sapiens)Dicer-/-;
Dicer-/- clone #45
Korean Collection for
Type Cultures (KCTC);
PMID: 26976607
KCTC: cat#HC19024HCT116 clone #45 with homozygous
knockout of Dicer protein; knockout achieved using
CRISPR/Cas9 which resulted in a 53 nucleotide
deletion in one allele and a 28 nucleotide
deletion in the other
Cell line (H. sapiens)293TATCCATCC: CRL-3216Derived from HEK293 cells (ATCC: CRL-1573);
express large T antigen; used for
packaging viruses
Cell line (H. sapiens)H460ATCCATCC: #HTB-177Human lung pleural effusion carcinoma
Cell line (Mus musculus)3LLATCCATCC #CRL-1642Mouse Lewis lung carcinoma
Cell line (Mus musculus)M565PMID: 25366259NAMouse hepatocellular carcinoma
isolated from naturally occurring tumor
in a floxed CD95 background
Antibodyanti-human AGO1
(rabbit monoclonal)
Cell SignalingCell Signaling #50531:2000; for western blot; primary Ab
Antibodyanti-human AGO1
(rabbit polyclonal)
AbcamAbcam #980561:2000; for western blot; primary Ab
Antibodyanti-human AGO2
(rabbit polyclonal)
AbcamAbcam #323811:500; for western blot; primary Ab
AntibodyGoat anti-rabbit,
IgG-HRP
Southern BiotechSouthern Biotech:
cat#SB-4030–05
1:5000; for western blot; secondary Ab
AntibodyAnti-Argonaute-2
antibody (rabbit
monoclonal)
[EPR10411]
AbcamAbcam #1867331:1200; for western blot; primary Ab
AntibodyAnti-Human CD178
antibody (Mouse IgG1)
Clone G247-4
BD PharmingenBD Pharmingen
#556387
1 μg/ml; for western blot; primary Ab
AntibodyAnti-CD95 (rabbit
polyclonal, C-20)
Santa CruzSanta Cruz #sc-715
(since discontinued)
1:1000; for western blot; primary Ab
AntibodyGoat anti-rabbit,
IgG-HRP
Cell SignalingCell Signaling: cat#70741:2000; for western blot; secondary Ab
AntibodyGoat anti-mouse;
IgG1-HRP
Southern BiotechSouthern BioTech:
cat#1070–05
1:5000; for western blot; secondary Ab
Recombinant
protein reagent
LzCD95LPMID: 14504390NALeucine zipper tagged CD95L;
recombinant protein
Chemical compoundCellTiter-GloPromegaPromega #G7570Detects ATP release as a surrogate
for cell death; read-out is fluorescence
Chemical compoundpropidium iodideSigma-AldrichSigma-Aldrich:
cat#P4864
Used for subG1 flow cytometry analysis
Chemical compoundpuromycinSigma-AldrichSigma-Aldrich:
cat#P9620
Used for selection of cells expressing
puromycin resistance cassettes
Chemical compound2',7'-dichlorodihydrofluorescein diacetateThermofisher ScientificThermofisher
Scientific #D399
Dye used for detecting ROS production
Chemical compoundzVAD-fmkSigma-AldrichSigma-Aldrich:
cat#V116
Used at 20 uM; pan caspase inhibitor
Recombinant
DNA reagent
pLenti-GIII-CMV-
RFP-2A-Puro
vector; pLenti
ABM IncNApLenti control empty lentiviral vector;
carries an RFP-2a-puromycin
resistance cassette
Recombinant
DNA reagent
pLenti-CD95Lthis paperNApLenti-GIII-CMV-RFP-2A-Puro vector that
expresses human wild type CD95L cDNA
(NM_000639.2); used to express wt human
CD95L upon infection with lentiviral particles
Recombinant
DNA reagent
pLenti-CD95LMUTthis paperNApLenti-GIII-CMV-RFP-2A-Puro vector that
expresses human CD95L cDNA
(NM_000639.2) with two nucleotide
substitutions in codon 218 (TAT - > CGT)
resulting in replacement of tyrosine
for arginine (Y218R mutation);
unable to bind CD95
Recombinant
DNA reagent
pLenti-CD95LMUTNPthis paperNApLenti-GIII-CMV-RFP-2A-Puro vector that
expresses human CD95L cDNA (NM_000639.2)
with both the Y218R mutation and a
single nucleotide substitution at the
second codon (CAG - > TAG), resulting in a
premature stop codon right
after the start codon
Recombinant
DNA reagent
pLenti-CD95LSILthis paperNApLenti-GIII-CMV-RFP-2A-Puro vector that
expresses human CD95L cDNA (NM_000639.2)
with all codons containing synonymous
mutations except for select codons in the
proline-rich domain to meet
IDT synthesis criteria
Transfected
construct
gRNA scaffoldPMID: 23287722IDT: synthesized
as gene block
455 nucleotide CRISPR/Cas9 gRNA scaffold
synthesized as a gene block; contains
promoter, gRNA scaffold, target sequence,
and termination sequence; scaffold
transcribes gRNAs that target Cas9
endonuclease to cut at target sites;
target sequences consist of 19 nucleotides
that are complementary to the target
site of choice; co-transfected with
Cas9 to catalyze cleavage.
Recombinant
DNA reagent
pLenti-CD95LMUTNP
(G258A)
This paperNApLenti-GIII-CMV-RFP-2A-Puro vector that
expresses human CD95L cDNA
(NM_000639.2) with the Y218R mutation,
and two additional single nucleotide
substitutions; one at the second codon
(CAG - > TAG), resulting in a premature stop
codon right after the start codon, and
another, G258A, resulting in the replacement
of a methionine with an isoleucine, thus
removing the alternative
translational start site.
Transfected
construct
pMJ920 Cas9 plasmidAddgene;
PMID: 23386978
Addgene: cat#42234Plasmid that expresses a human
codon-optimized Cas9 tagged with
GFP and HA; used to express Cas9 for
CRISPR-mediated deletions.
Chemical
compound
Lipofectamine 2000ThermoFisher ScientificThermoFisher
Scientific:
cat#11668019
Transfection reagent
Chemical
compound
Lipofectamine RNAiMAXThermoFisher ScientificThermoFisher
Scientific:
cat#13778150
Transfection reagent; used for
transfection of small
RNAs such as siRNAs
Commercial
assay or kit
StrataClone Blunt
PCR Cloning Kit
Agilent TechnologiesAgilent
Technologies:
cat#240207
Used to blunt-end clone the gRNA
scaffolds into the pSC-B plasmid
Genetic reagentTaqman Gene
expression master mix
ThermoFisher Scientific#4369016
Sequence-based
reagent
shR6 flanking Fr primerIDTIDT: custom
DNA oligo
Fr primer that flanks shR6 site;
used to detect 227 nt shR6 deletion;
5’-GGTGTCATGCTGTGACTGTTG-3’
Sequence-based
reagent
shR6 flanking Rev primerIDTIDT: custom
DNA oligo
Rev primer that flanks shR6 site;
used to detect 227 nt shR6 deletion;
5’-TTTAGCTTAAGTGGCCAGCAA-3’
Sequence-based
reagent
shR6 internal Rev primerIDTIDT: custom
DNA oligo
Rev primer that overlaps
with the shR6 site;
used to detect 227 nt shR6 deletion;
5’-AAGTTGGTTTACATCTGCAC-3’
Sequence-based
reagent
CD95 flanking Fr primerIDTIDT: custom
DNA oligo
Fr primer that flanks the CD95 gene;
used to detect CD95 gene deletion;
5’-TGTTTAATATAGCTGGGGCTATGC-3'
Sequence-based
reagent
CD95 flanking Rev primerIDTIDT: custom
DNA oligo
Rev primer that flanks the CD95 gene;
used to detect CD95 gene deletion;
5’-TGGGACTCATGGGTTAAATAGAAT-3’
Sequence-based
reagent
CD95 internal Rev primerIDTIDT: custom
DNA oligo
Rev internal primer that targets within
the CD95 gene; used to detect CD95
gene deletion;
5’-GACCAGTCTTCTCATTTCAGAGGT-3’
Sequence-based
reagent
siScr/siNT1IDT;IDT: custom
DNA oligo
control non-targeting siRNA;
sense: UGGUUUACAUGUCGACUAA-3'
Sequence-based
reagent
c7/1IDTcustom siRNA;
antisense strand
corresponds to
cluster 7 CD95L
sequence
antisense: 5’-AUUGGGCCUG
GGGAUGUUU-3';
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at
the first two positions
Sequence-based
reagent
c7/2IDTcustom siRNA;
antisense strand
corresponds to
cluster 7 CD95L
sequence
antisense: 5’-CCUGGGGAU
GUUUCAGCUC-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at the
first two positions
Sequence-based
reagent
c11IDTcustom siRNA;
antisense strand
corresponds to
cluster 11 CD95L
sequence
antisense: 5’-CCAACUCAAGG
UCCAUGCC-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at the
first two positions
Sequence-based
reagent
c15/1IDTcustom siRNA;
antisense strand
corresponds to
cluster 15 CD95L
sequence
antisense: 5’-AAACUGGGCUGU
ACUUUGU-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at
the first two positions
Sequence-based
reagent
c15/2IDTcustom siRNA;
antisense strand
corresponds to
cluster 15 CD95L
sequence
antisense: 5’- AACUGGGCUGU
ACUUUGUA-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at the
first two positions
Sequence-based
reagent
c16/1IDTcustom siRNA;
antisense strand
corresponds to
cluster 16 CD95L
sequence
antisense: 5’- CAACAACCUGCC
CCUGAGC-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at the
first two positions
Sequence-based
reagent
c16/2IDTcustom siRNA;
antisense strand
corresponds to
cluster 16 CD95L
sequence
antisense: 5’- AACUCUAAGCG
UCCCCAGG-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at
the first two positions
Sequence-based
reagent
c21IDTcustom siRNA;
antisense strand
corresponds to
cluster 21 CD95L
sequence
antisense: 5’- UCAACGUAUC
UGAGCUCUC-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at the
first two positions
Sequence-based
reagent
c22IDTcustom siRNA;
antisense strand
corresponds to
cluster 22 CD95L
sequence
antisense: 5’- AAUCUCAGACG
UUUUUCGG-3’;
antisense strand designed with 3'
deoxy AA; complementary sense strand
has 3' deoxy TT and 2'-O-methylation at
the first two positions
Sequence-based
reagent
siCtr poolDharmaconD-001810–10control non-targeting siRNA pool
Sequence-based
reagent
SMARTpool siRNA
targeting AGO2
DharmaconL-004639-00-0005siRNA pool designed to target AGO2
Sequence based
reagent (human)
GAPDH primerThermofisher ScientificHs00266705_g1RT-qPCR; control probe
Sequence based
reagent (human)
CD95L primersThermofisher ScientificHs00181226_g1;
Hs00181225_m1
RT-qPCR
Sequence based
reagent (human)
CD95 primersThermofisher ScientificHs00531110_m1;
Hs00236330_m1
RT-qPCR
Sequence based
reagent (human)
CD95LSIL primerThermofisher Scientificassay ID:
APNKTUD
Custom RT-qPCR primer designed
using the Thermofisher Scientific
design tool to detect CD95LSIL mRNA
Sequence based
reagent (human)
Cluster 8 CD95L
small RNA primer
Thermofisher Scientificcustom probe
Assay ID:
CT7DPEM
Custom RT-qPCR primer designed using
the Thermofisher Scientific design tool at
https://www.thermofisher.com/order/custom-
genomic-products/tools/small-rna to
specifically detect small RNAs from
cluster 8 of CD95L
(5’- AAGGAGCTGGCAGAACTCCGAGA-3’)
Sequence based
reagent (human)
Cluster 21 CD95L
small RNA primer
Thermofisher Scientificcustom probe
Assay ID:
CTAAADA
Custom RT-qPCR primer
designed using the Thermofisher
Scientific design tool at
https://www.thermofisher.com/order/custom-
genomic-products/tools/small-rna to
specifically detect small RNAs from cluster 21
of CD95L
(5’- TCAACGTATCTGAGCTCTCTC-3’)
Sequence based
reagent (human)
z30 primerThermofisher ScientificThermoFisher
Scientific #4427975
RT-qPCR for small RNA;
control probe
Peptide,
recombinant
protein
Flag-GST-T6B
peptide
PMID: 26351695NAPeptide derived from
GW182/TNRC6B used to
pull down AGO1 to 4
Commercial
assay or kit
anti-Flag M2
magnetic beads
Sigma-AldrichSigma-Aldrich #M8823

Data availability

Sequencing data have been deposited in GEO under accession codes: GSE103631 and GSE114425.

The following data sets were generated
  1. 1
    CD95/Fas ligand mRNA is toxic to cells
    1. Putzbach W
    2. Peter ME
    3. Bartom E
    (2018)
    Publicly available at the NCBI Gene Expression Omnibus (accession no. GSE103631).
  2. 2
    CD95L mRNA is toxic to cells
    1. Putzbach WE
    2. Haluck-Kangas A
    3. Gao QQ
    4. Sarshad AA
    5. Bartom E
    6. Stults A
    7. Qadir AS
    8. Scholtens DM
    9. Hafner M
    10. Peter ME
    (2018)
    Publicly available at the NCBI Gene Expression Omnibus (accession no. GSE114425).

Additional files

Supplementary file 1

Reads from coding and noncoding genes pulled-down with Ago proteins in HCT116 Drosha k.o. pLenti-CD95L cells.

Tab 1: Reads from all genes; Tab 2: Reads from processed protein coding genes (>10 reads), Tab 3: Reads from unprocessed protein coding genes (>10 reads).

https://doi.org/10.7554/eLife.38621.020
Transparent reporting form
https://doi.org/10.7554/eLife.38621.021

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