Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins
- Stanford University, United States
- Harvard University, United States
- Brown University, United States
- Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), United States
Figures
Figure 1 with 1 supplement
Single and paired-gene CRISPRi screens implicate TRC pathway inhibition as the MOA of Retro-2.
(A) Schematic of single-gene CRISPRi screen. A 288 gene library with 10 guides per gene targeting previously identified ricin hits and 2000 negative controls was lentivirally infected into a K562 …
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Figure 1—source data 1
Sequencing counts for single gene screens.
- https://doi.org/10.7554/eLife.48434.004
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Figure 1—source data 2
Results for single gene screens.
- https://doi.org/10.7554/eLife.48434.005
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Figure 1—source data 3
Sequencing counts for double gene screens.
- https://doi.org/10.7554/eLife.48434.006
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Figure 1—source data 4
Results for double gene screens.
- https://doi.org/10.7554/eLife.48434.007
Figure 1—figure supplement 1
Genetic profile analysis of ricin phenotypes for Retro-2 and candidate genes.
(A) Reproducibility of genes’ knockdown effect on ricin resistance in the presence of Retro-2 for the CRIPSRi single-gene screen. The value is calculated by comparing the enrichment of guides …
Figure 2 with 1 supplement
Retro-2 diverts newly-synthesized TA proteins from ER targeting to degradation.
(A) Schematic of the dual-colour reporter consisting of a self-cleaving P2A peptide between a GFP and a RFP with a C-terminal TMD. Genetic and chemical perturbations to targeting pathways will …
Figure 2—figure supplement 1
ASNA1 knockout and Retro-2/DHQZ36.1 treatment destabilize the transmembrane domain of STX5.
(A) Left: Whole-cell lysates were prepared from wildtype and ASNA1KO HEK293T cells and subjected to SDS-PAGE and visualized by immunoblotting (IB). SQSTM1 was used as the loading control. ASNA1KO …
Figure 3 with 1 supplement
ASNA1 knockdown and Retro-2 treatment both decrease the abundance of Golgi-localized STX5, an ASNA1 substrate.
(A) HeLa cells expressing either ASNA1-targeting or scrambled control (shCtrl) shRNAs were treated for 24 hr with DMSO or 10 μM Retro-2 before fixation and staining for STX5, a Golgi marker (GM130), …
Figure 3—figure supplement 1
Effect of ASNA1 knockdown or Retro-2 and DHQZ36.1 in HeLa cells.
(A) Dose curves for the protective effect of Retro-2 against ricin cytotoxicity in HeLa cells expressing either shRNAs targeting ASNA1 or scrambled negative controls. Cells are pretreated with the …
Figure 4 with 2 supplements
Isolation and characterization of A149V ASNA1, a Retro-2-resistance allele.
(A) Schematic of ASNA1 mutagenesis by CRISPR-X. A 172 sgRNA library tiling the ASNA1 coding region was lentivirally infected in a K562 cell line expressing a dCas9-AID*Δ N-terminal fusion. The pool …
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Figure 4—source data 1
Results for CRISPR-X experiments to identify ASNA-1 mutations resistant to Retro-2.
- https://doi.org/10.7554/eLife.48434.015
Figure 4—figure supplement 1
Replication and validation of CRISPR-X screen.
(A) Allelic fraction of ASNA1 mutations post-selection for second replicate. The frequency of mutations across the gene-body of ASNA1 in the second replicate of the selected population. Top mutation …
Figure 4—figure supplement 2
Characterization of clonal ASNA1 A149V cells.
(A) Clonal K562 lines containing the mutant A149V (C460T), synonymous A149A (C461T), and wildtype ASNA1 alleles were generated by single cell sorting of edited pools. Homozygous ASNA1 alleles were …
Figure 5 with 2 supplements
DHQZ36.1 blocks substrate transfer from SGTA to ASNA1 in vitro.
(A) Schematic of the fractionation and crosslinking assay to monitor the cytosolic interactions of in vitro translated TA proteins. After translation in crude rabbit reticulate lysate (RRL), the TA …
Figure 5—figure supplement 1
In vitro analysis of DHQZ36.1’s effect on ER targeting.
(A) Crude RRL lysate was size-separated on a 5–25% sucrose gradient. The fractions were subjected to SDS-PAGE and visualized by immunoblotting for BAG6, ASNA1, SGTA, and GET4. (B) Uncropped version …
Figure 5—figure supplement 2
Biochemical analysis of mutant ASNA1.
(A) Clustal Omega amino acid sequence alignment of ASNA1 homologs from Homo Sapiens (NP_004308.2) and Danio rerio (NP_001002298.1) and Get3 from Saccharomyces cerevisiae (NP_010183.1). Highlighted …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.48434.019
