Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Scatter plot showing the relationship between changes in RNA-binding protein (RBP) recruitment to poly(A)-tailed mRNAs at 18 hpi with SINV (y-axis) and changes in their recruitment to ribosomes (x-axis). (B) Volcano plot showing changes in polysome-associated proteins at 12 hpi with SINV. RBPs previously identified as specifically binding to viral RNAs are colored burgundy. DEAD-box and DExH-box helicases, as well as ASCC2 and ASCC3, which also bind viral RNAs, are highlighted in green and turquoise, respectively. Viral proteins are not shown on the volcano plot.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: RNA Binding Assay, Binding Assay

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Measurement of mCherry fluorescence serves as an indicator of SINV replication in cells that were transfected with the indicated sgRNA to edit genes encoding the different components of the ribosome-associated quality control-trigger (RQT) complex. The cells were infected at a low MOI for 24 hours. The bar plot shows the mean of three biological replicates, with each replicate consisting of three to four technical replicates. The error bars represent the standard error of the mean. Two-tailed paired t-tests were performed to compare the mCherry signal in individual knockout cells to the signal in control knockout cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. “ns” indicates not significant. (B) Immunoblots showing the efficiency with which each subunit of the RQT complex was knocked out. The β-actin immunoblot serves as a loading control. (C) Cells were either uninfected or infected with SINV at a high MOI for 12 hours. Before collection, the cells were left untreated or treated with 2 µM of anisomycin for 15 minutes, and lysed in RIPA buffer supplemented with 10 mM N-Ethylmaleimide. Cell lysates were used for immunoblotting with antibodies against the ribosomal proteins eS10, uS10, uS3, and uS5. The ubiquitin-modified ribosomal proteins are indicated on the blots as brown colors. (D) Cells were transfected with either a control sgRNA or one targeting PKR. Then, the cells were infected, treated with anisomycin, and lysed as described in panel C. An immunoblot against eS10 is shown, with the ubiquitinated eS10 indicated. (E) Schematic representation of the signaling pathways potentially activated in response to ribosome collisions, along with their corresponding collided ribosome sensors. (F) A bar plot showing the quantification of mCherry fluorescence in cells transfected with the indicated sgRNAs for gene editing and then infected with SINV at a low MOI for 24 hours. The bar plot represents the mean of three biological replicates, each consisting of three to four technical replicates. Error bars and statistical tests are as described in Panel A. (G) Immunoblot analysis of SINV capsid protein levels in cells transfected with ZNF598 - or GCN1 -targeting sgRNAs. Two different sgRNAs were tested for GCN1 gene editing. The cells were infected at a high MOI for 13 hours. The immunoblots below show the efficiency of ZNF598 or GCN1 gene knockout, with the β-actin immunoblots serving as loading controls. (H) Immunoblot analysis of ZAKα-mediated p38 phosphorylation in cells infected with SINV at a high MOI for 13 hours. UV-C treatment was used as a positive control for phosphorylated p38. (I) Schematic representation of the ubiquitination and de-ubiquitination of uS3 and uS5 by RNF10 and USP10, respectively, in response to ribosome stalling during translation initiation and elongation. (J) Bar plot showing mCherry fluorescence quantification in cells transfected with the indicated sgRNAs and subsequently infected with SINV at a low MOI for 24 hours. This bar plot represents the mean of two or three biological replicates, with each replicate consisting of three to four technical replicates. The error bars and statistical tests are as described in Panel A. (K) Immunoblot analysis of SINV capsid protein levels in cells transfected with either RNF10 - or USP10 -targeting sgRNAs. The cells were infected at a high MOI for 12 hours. Immunoblots against uS3 and uS5 validate the effectiveness of the RNF10 and USP10 knockouts by revealing decreased and increased levels of the uS3/uS5 ubiquitinated forms, respectively. The β-actin immunoblot serves as a loading control. (L) Immunoblot analysis of uS3 ubiquitination in cells transfected with sgRNAs targeting either ASCC3 or ZNF598 . The cells were infected at a high MOI for 12 hours.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: Fluorescence, Transfection, Control, Infection, Two Tailed Test, Knock-Out, Western Blot, Ubiquitin Proteomics, Modification, Protein-Protein interactions, Gene Knockout, Phospho-proteomics, Positive Control

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Functional enrichment analysis of ASCC3 -knockout cells using the STRING database . (B) Volcano plot showing changes in protein abundance in ASCC3 -knockout cells compared to control knockout cells. Proteins containing a signal peptide are colored turquoise and ASCC3 is colored fuchsia. (C) Scatterplot showing the relationship between changes in protein abundance in ZNF598 -knockout cells (x-axis) and ASCC3 -knockout cells (y-axis). Proteins containing a signal peptide are colored turquoise. ZNF598, ASCC3, and ASCC2 are colored fuchsia. (D) Quantification of all SINV proteins in cells lacking ASCC3 by mass spectrometry, compared to control cells. The cells were infected at a high MOI for 13 hours. (E) Cell lysates from control- and ASCC3 -knockout cells were separated by polyacrylamide gel electrophoresis. The glycoproteins were stained with Pro-Q Emerald 488 dye, and the resulting green fluorescent signals were imaged (see upper panel). Total proteins were subsequently stained with Coomassie Blue (lower panel). The bar plot shows the quantification of glycoprotein signal intensity and represents the mean of three biological replicates. (F) Control and ASCC3 -knockout cells were subjected to subcellular fractionation of the cytosol and membranes. An immunoblot was performed to analyze the subcellular localization of ASCC3. The quality of the cell fractionation was verified by immunoblotting the ER proteins STT3A and SRPRA. The β-actin immunoblot serves as a loading control. (G) Cells that were either left uninfected or infected with SINV at a high MOI for 13 hours were subjected to subcellular fractionation of the cytosol and membranes. An immunoblot was performed to analyze the subcellular localization of ASCC3 in response to viral infection. An immunoblot against ubiquitinated uS3 was performed to evaluate the partitioning of stalled ribosomes between the cytosol and the ER. The quality of the cell fractionation was verified by immunoblotting the ER protein DDRGK1.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: Functional Assay, Knock-Out, Quantitative Proteomics, Control, Mass Spectrometry, Infection, Polyacrylamide Gel Electrophoresis, Staining, Fractionation, Western Blot, Cell Fractionation

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Representative images showing the localization of ASCC3 and SINV subgenomic RNA by combined immunofluorescence and in situ hybridization in HEK293T cells. Control and ASCC3 -knockout cells were either mock-infected or infected with SINV for 12 hours. (B) Fluorescence intensities of ASCC3, SINV RNA, and nuclear signals measured in over 90 cells shown in panel (A). (C) Correlation between ASCC3 and SINV RNA fluorescence signals in infected control cells, with a calculated Pearson coefficient of R = 0.66.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: Immunofluorescence, In Situ Hybridization, Control, Knock-Out, Infection, Fluorescence

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Heatmaps of gene-level sgRNA log 2 FC for 16 genes selected for validation; white: not significant (two-sided Mann–Whitney test P > 0.1). b , Schematic of individual sgRNA expression construct. Puro: puromycin resistance gene. c , Correlation of single sgRNA phenotype from growth competition assays [log 2 (-/+Dox)] and log 2 FC of the same sgRNA in the pooled CRISPRi screen (Spearman’s R, P < 0.05) for the two most active sgRNAs (sgRNA1 and sgRNA2) for each gene in the screens. Solid lines denote linear regression model; grey shading denotes 95% confidence interval. d , Knockdown efficiency of genes from ( a ) compared to a non-targeting control (sgControl) measured by quantitative RT-PCR for the most active sgRNA from the screens (sgRNA1; n = 2 biological replicates, each with n = 3 technical replicates). e , Correlation plot of cell type specificity (hiPSCi log 2 FC minus HEK293i log 2 FC) calculated for individual sgRNAs from growth competition assays [∆log 2 FC (validation), n = 2 biological replicates] and the same sgRNAs in the pooled CRISPRi screen [∆log 2 FC (screen); n = 3 biological replicates]. Solid line denotes linear regression model; grey shading denotes 95% confidence interval. f , Heatmaps of significant (pairwise two-tailed t-test, FDR < 0.01) protein-level log 2 FC for screening targets in ( a ) measured by mass spectrometry in the indicated cell types in comparison to hiPSCi ( n = 3 biological replicates; numbers in brackets indicate isoforms). g , Immunoblot analysis of ZNF598, ASCC3, HBS1L, and PELO in sgRNA-transduced cells after KRAB-dCas9 induction ( + Dox) and in matched uninduced controls (-Dox).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Biomarker Discovery, MANN-WHITNEY, Expressing, Construct, Knockdown, Control, Quantitative RT-PCR, Two Tailed Test, Mass Spectrometry, Comparison, Western Blot

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a,b , Growth assays of HEK293i or hiPSCi expressing a sgRNA targeting N4BP2 ( a ) or EDF1 ( b ) ( n = 2 biological replicates; line: average) and knockdown efficiency measurements of target genes compared to a non-targeting control (sgControl) by quantitative RT-PCR ( n = 2 biological replicates, each with n = 3 technical replicates). c , Growth assays of cells transduced with the second most potent sgRNA (sgRNA2) targeting ZNF598 , ASCC3 , HBS1L , or PELO in the hiPSCi screen ( n = 3 biological replicates; line: average). The percentage of GFP-positive (GFP + ) cells was measured by flow cytometry ( > 10,000 cells/ analysis) every three population doublings and normalized to GFP+ cell numbers in matched uninduced (-Dox) controls. d , Growth assays of hiPSCi transduced with a sgRNA targeting HBS1L alone or in combination with a cDNA encoding wild-type or a GTPase-inactive HBS1L (H348A) ( n = 3 biological replicates, P -values from unpaired two-tailed t-test). e , Heatmaps of significant (pairwise two-tailed t-test, FDR < 0.01) protein-level log 2 FC for SKI complex components in the indicated cell types in comparison to hiPSCi measured by mass spectrometry ( n = 3 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Knockdown, Control, Quantitative RT-PCR, Transduction, Flow Cytometry, Two Tailed Test, Comparison, Mass Spectrometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Schematic of expression constructs and workflows for growth competition and stalling reporter readthrough assays. b , Growth assays of cells transduced with the most potent sgRNA targeting ZNF598 , ASCC3 , HBS1L or PELO in the inducible hiPS cell screen ( n = 2 biological replicates; line, mean). The percentage of GFP-positive (GFP + ) cells was measured by flow cytometry (>10,000 cells per analysis) every four population doublings and normalized to GFP + cell numbers in matched uninduced (−Dox) controls. GTP, guanosine triphosphate; GDP, guanosine diphosphate. c – e , Stalling readthrough of reporters containing (AAA) 20 ( c ), the XBP1 arrest peptide ( d ) and a no-stall control ( e ). The median fluorescence intensity for BFP and mOrange was quantified by flow cytometry (>20,000 cells per analysis) in the indicated cell lines transduced with the most potent sgRNA targeting ZNF598 , ASCC3 , HBS1L or PELO based on the inducible hiPS cell screen. The ratio of mOrange to BFP in knockdown cells was normalized to that for the same reporter in cells transduced with a nontargeting sgRNA (sgControl; n = 3 biological replicates; P values from an unpaired two-tailed t -test). UbC, ubiquitin C promoter; FLAG, Flag-tag.

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Construct, Transduction, Flow Cytometry, Control, Fluorescence, Knockdown, Two Tailed Test, Ubiquitin Proteomics, FLAG-tag

Journal: Journal of Translational Medicine

Article Title: ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA

doi: 10.1186/s12967-021-02948-6

Figure Lengend Snippet: MMS-induced P-bodies in HeLa are enriched in ALKBH3 and localized adjacent to ASCC3. A HeLa cells transfected with P-body marker CFP-DCP1A were treated for 1 h as indicated, fixed, and double stained with anti-ASCC3 (red) and anti-ALKBH3 (green). B Enlarged images from white squares in A showing overlap of DCP1A and ALKBH3, and accumulation of ASCC3 at the P-body periphery (upper panel, corresponding to section 1). The lower panel shows z-stacks of section 2 demonstrating that ASCC3 accumulates around MMS-induced P-bodies whereas ALKBH3 accumulates inside the same P-bodies (lower panel)

Article Snippet: Primary antibodies and dilutions: ALKBH3 (Santa Cruz, sc-376520, IF: 1/100, WB: 1/2000), ASCC3 (Atlas, HPA001439, IF: 1/50), β -actin (Abcam, ab8226, IB: 1/2000), CELF1 (Santa Cruz, SC-20003, IB: 1/1000, IF: 1/200), EIF2S1 (Atlas, HPA064885, IB: 1/1000), EIF2S1-pS51 [Abcam, ab32157, IB: 1/500.

Techniques: Transfection, Marker, Staining

Journal: Journal of Translational Medicine

Article Title: ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA

doi: 10.1186/s12967-021-02948-6

Figure Lengend Snippet: ASCC3 promotes formation of P-bodies in PC-3 cells. PC-3 cells transfected with P-body marker CFP-DCP1A were MMS-treated for 1 h as indicated, fixed, and double stained with anti-ASCC3 (red) and anti-ALKBH3 (green). A PC-3 cells proficient in ASCC3 contain P-bodies both in the absence (upper panel) and presence (middle panel) of MMS-treatment. The bottom panel shows enlarged z-stack demonstrating co-localization of DCP1A and ALKBH3, whereas the P-body interior contains less ASCC3 than the volume immediately surrounding the same P-body. B Formation of P-bodies was severely impeded in ASCC3 knockdown PC-3 cells both in the presence and absence of MMS. C Quantitative analysis of P-bodies in WT and ASCC3 knockdown (Kd) PC-3 after 2 mM MMS treatment demonstrated that significantly fewer ASCC3-deficient cells contained P-bodies compared to WT. In the cells that contained P-bodies, these were also significantly fewer and smaller than in the parental cells

Article Snippet: Primary antibodies and dilutions: ALKBH3 (Santa Cruz, sc-376520, IF: 1/100, WB: 1/2000), ASCC3 (Atlas, HPA001439, IF: 1/50), β -actin (Abcam, ab8226, IB: 1/2000), CELF1 (Santa Cruz, SC-20003, IB: 1/1000, IF: 1/200), EIF2S1 (Atlas, HPA064885, IB: 1/1000), EIF2S1-pS51 [Abcam, ab32157, IB: 1/500.

Techniques: Transfection, Marker, Staining, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA

doi: 10.1186/s12967-021-02948-6

Figure Lengend Snippet: Working model of cellular processing of aberrantly (MMS-) methylated mRNA. MMS-induced m 1 A and m 3 C mediate direct ribosomal stalling, whereas m 7 G may form translation-blocking secondary structures. Collided disomes are detected by ZNF598, which serves two functions: It stabilizes GIGYF2-4EHP to block cap-dependent 43S entry and facilitates recruitment of ASCC3/ALKBH3 to mediate ribosome splitting and removal of m 1 A/m 3 C. Repaired transcripts can then be re-routed directly to translation. Transcripts harboring persistent blocking lesions, e.g. MMS-induced secondary structures, are stripped of ribosomes by ASCC3 and accumulate in P-bodies. Here, helicase activities relieve secondary structures to promote repair by ALBH3, whereas nonrepairable mRNAs are degraded

Article Snippet: Primary antibodies and dilutions: ALKBH3 (Santa Cruz, sc-376520, IF: 1/100, WB: 1/2000), ASCC3 (Atlas, HPA001439, IF: 1/50), β -actin (Abcam, ab8226, IB: 1/2000), CELF1 (Santa Cruz, SC-20003, IB: 1/1000, IF: 1/200), EIF2S1 (Atlas, HPA064885, IB: 1/1000), EIF2S1-pS51 [Abcam, ab32157, IB: 1/500.

Techniques: Methylation, Blocking Assay

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Heatmaps of gene-level sgRNA log 2 FC for 16 genes selected for validation; white: not significant (two-sided Mann–Whitney test P > 0.1). b , Schematic of individual sgRNA expression construct. Puro: puromycin resistance gene. c , Correlation of single sgRNA phenotype from growth competition assays [log 2 (-/+Dox)] and log 2 FC of the same sgRNA in the pooled CRISPRi screen (Spearman’s R, P < 0.05) for the two most active sgRNAs (sgRNA1 and sgRNA2) for each gene in the screens. Solid lines denote linear regression model; grey shading denotes 95% confidence interval. d , Knockdown efficiency of genes from ( a ) compared to a non-targeting control (sgControl) measured by quantitative RT-PCR for the most active sgRNA from the screens (sgRNA1; n = 2 biological replicates, each with n = 3 technical replicates). e , Correlation plot of cell type specificity (hiPSCi log 2 FC minus HEK293i log 2 FC) calculated for individual sgRNAs from growth competition assays [∆log 2 FC (validation), n = 2 biological replicates] and the same sgRNAs in the pooled CRISPRi screen [∆log 2 FC (screen); n = 3 biological replicates]. Solid line denotes linear regression model; grey shading denotes 95% confidence interval. f , Heatmaps of significant (pairwise two-tailed t-test, FDR < 0.01) protein-level log 2 FC for screening targets in ( a ) measured by mass spectrometry in the indicated cell types in comparison to hiPSCi ( n = 3 biological replicates; numbers in brackets indicate isoforms). g , Immunoblot analysis of ZNF598, ASCC3, HBS1L, and PELO in sgRNA-transduced cells after KRAB-dCas9 induction ( + Dox) and in matched uninduced controls (-Dox).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Biomarker Discovery, MANN-WHITNEY, Expressing, Construct, Knockdown, Control, Quantitative RT-PCR, Two Tailed Test, Mass Spectrometry, Comparison, Western Blot

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a,b , Growth assays of HEK293i or hiPSCi expressing a sgRNA targeting N4BP2 ( a ) or EDF1 ( b ) ( n = 2 biological replicates; line: average) and knockdown efficiency measurements of target genes compared to a non-targeting control (sgControl) by quantitative RT-PCR ( n = 2 biological replicates, each with n = 3 technical replicates). c , Growth assays of cells transduced with the second most potent sgRNA (sgRNA2) targeting ZNF598 , ASCC3 , HBS1L , or PELO in the hiPSCi screen ( n = 3 biological replicates; line: average). The percentage of GFP-positive (GFP + ) cells was measured by flow cytometry ( > 10,000 cells/ analysis) every three population doublings and normalized to GFP+ cell numbers in matched uninduced (-Dox) controls. d , Growth assays of hiPSCi transduced with a sgRNA targeting HBS1L alone or in combination with a cDNA encoding wild-type or a GTPase-inactive HBS1L (H348A) ( n = 3 biological replicates, P -values from unpaired two-tailed t-test). e , Heatmaps of significant (pairwise two-tailed t-test, FDR < 0.01) protein-level log 2 FC for SKI complex components in the indicated cell types in comparison to hiPSCi measured by mass spectrometry ( n = 3 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Knockdown, Control, Quantitative RT-PCR, Transduction, Flow Cytometry, Two Tailed Test, Comparison, Mass Spectrometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Schematic of expression constructs and workflows for growth competition and stalling reporter readthrough assays. b , Growth assays of cells transduced with the most potent sgRNA targeting ZNF598 , ASCC3 , HBS1L or PELO in the inducible hiPS cell screen ( n = 2 biological replicates; line, mean). The percentage of GFP-positive (GFP + ) cells was measured by flow cytometry (>10,000 cells per analysis) every four population doublings and normalized to GFP + cell numbers in matched uninduced (−Dox) controls. GTP, guanosine triphosphate; GDP, guanosine diphosphate. c – e , Stalling readthrough of reporters containing (AAA) 20 ( c ), the XBP1 arrest peptide ( d ) and a no-stall control ( e ). The median fluorescence intensity for BFP and mOrange was quantified by flow cytometry (>20,000 cells per analysis) in the indicated cell lines transduced with the most potent sgRNA targeting ZNF598 , ASCC3 , HBS1L or PELO based on the inducible hiPS cell screen. The ratio of mOrange to BFP in knockdown cells was normalized to that for the same reporter in cells transduced with a nontargeting sgRNA (sgControl; n = 3 biological replicates; P values from an unpaired two-tailed t -test). UbC, ubiquitin C promoter; FLAG, Flag-tag.

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Construct, Transduction, Flow Cytometry, Control, Fluorescence, Knockdown, Two Tailed Test, Ubiquitin Proteomics, FLAG-tag

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Growth assays of cells transduced with a sgRNA targeting EIF2S1 in HEK293i, hiPSCi, or NPC cells ( n = 2 biological replicates; line: average) and knockdown efficiency measurements of target genes compared to a non-targeting control (sgControl) by quantitative RT-PCR ( n = 2 biological replicates, each with n = 3 technical replicates). b , Venn diagrams of overlap between genes upregulated or downregulated in HEK293i or hiPSCi depleted for ZNF598 or HBS1L by CRISPRi ( p -values from one-tailed Fisher’s exact test). c,d , Gene ontology (GO) enrichment in differentially expressed mRNAs using the “Biological Process” function in ClusterProfiler (one-tailed Fisher’s exact test with Benjamini-Hochberg correction, P-adj ≤ 0.01) in knockdown hiPSCi ( b ) or HEK293i ( c ). e, f , Flow cytometry analysis of eIF2α phosphorylation ( d ) and p38 phosphorylation ( e ) upon depletion of ZNF598 or HBS1L in hiPSCi in comparison to cells transduced with a non-targeting sgRNA (sgControl; n = 3 biological replicates; matched samples). hiPSC treated with 2.5 µg tunicamycin (TM) for 2 hours to induce the ISR or with 0.05 mg/l anisomycin (ANS) for 15 minutes to induce the RSR served as a positive control. g , Growth assays of hiPSCi expressing a non-targeting sgRNA (sgControl, day 6) in the absence (-) or presence of inhibitors of GCN2 (GCN2i, A-92, 1.25 µM), PERK (PERKi, GSK2606414, 4 nM), p38 (p38i, SB203580, 1 µM), or the ISR (ISRIB, 50 nM) in comparison to uninduced controls ( n = 3 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Transduction, Knockdown, Control, Quantitative RT-PCR, One-tailed Test, Flow Cytometry, Phospho-proteomics, Comparison, Positive Control, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Global protein synthesis measurements in knockdown inducible hiPS cells or inducible HEK293 cells by HPG labeling for 30 min. Median fluorescence intensity was quantified by flow cytometry (>10,000 cells per analysis) and normalized to values from cells transduced with sgControl ( n = 5 biological replicates; P values from an unpaired two-tailed t -test). b , LDH measurements in culture supernatants from knockdown inducible hiPS cells or inducible HEK293 cells. Values were normalized to supernatant from cells transduced with sgControl ( n = 4 technical replicates and 4 biological replicates; P values from an unpaired two-sided t -test). c , Heatmap of mRNAs differentially expressed upon ZNF598 or HBS1L repression in inducible hiPS cells or inducible HEK293 cells ( n = 2 biological replicates; two-sided Wald test with Benjamini–Hochberg correction, adjusted P ≤ 0.05; n = 3165). d , Heatmap of a data subset from c showing genes upregulated downstream of ATF4 within the ISR or downstream of ATF6 or IRE1α upon ER protein folding perturbations . e , Growth assays of inducible hiPS cells expressing an sgRNA targeting ZNF598 (day 6) or HBS1L (day 8) in the absence (−) or presence of GCN2i (A-92, 1.25 µM), PERKi (GSK2606414, 4 nM), p38i (SB203580, 1 µM) or ISRIB (50 nM) in comparison to uninduced controls ( n = 3 biological replicates; P values from an unpaired two-tailed t -test). f , Growth assays of inducible hiPS cells expressing an sgRNA targeting ZNF598 , HBS1L , GCN2 and PERK alone or in combination in comparison to uninduced controls ( n = 5 biological replicates; P values from an unpaired two-tailed t -test). g , Immunoblot analysis of eIF2α phosphorylation, p38 phosphorylation and total eIF2α and p38 levels in inducible hiPS cells and inducible HEK293 cells ( n = 4 biological replicates). Inducible hiPS cells treated with 2.5 µM tunicamycin (TM) for 2 h served as a positive control for induction of eIF2α phosphorylation; inducible hiPS cells treated with 0.05 mg L −1 ANS for 15 min served as a positive control for induction of p38 phosphorylation. Rep, replicate. h , Quantification of signal intensity in g by densitometry ( P values from an unpaired two-tailed t -test).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Knockdown, Labeling, Fluorescence, Flow Cytometry, Transduction, Two Tailed Test, Expressing, Comparison, Western Blot, Phospho-proteomics, Positive Control

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Venn diagram of overlaps between 5´ TOP motif mRNAs ( n = 97) and transcripts with significantly increased A-site pauses in the first 5 codons in ZNF598 RING -expressing hiPSCi ( n = 704), and comparison of 5´ UTR lengths and TPM levels from RNA-Seq in both groups ( p -value from two-sided Wilcoxon test). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. b , Comparison of ribosome recruitment scores for 5´ UTRs quantified by direct analysis of ribosome targeting (DART) for histone mRNAs with start site pauses ( n = 29), other mRNAs with start site pauses ( n = 351), other well-translated mRNAs ( n = 1340), and other detectable mRNAs ( n = 3791). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range. c , Density heatmaps of 50-80 nt ribosome footprints according to length and 5’ end position around CDS start (left) and stop sites (right) for a second biological replicate. d , Density heatmaps of 50-80 nt ribosome footprints according to length and 3’ end position around CDS start (left) and stop sites (right) for a second biological replicate. e , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in hiPSCi ( n = 2 biological replicates). f , Polysome profiles and immunoblot analysis of uS10 and eS10 in sucrose gradient fractions from control HEK293i or after a short (2.5 min) treatment with 2 µg/ml homoharringtonine (HAR) ( n = 1 biological replicate). g , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in ZNF598-depleted hiPSCi treated with GCN2 inhibitor (GCN2i, A-92, 1.25 µM ; n = 2 biological replicates). h , Polysome profiles (from Fig. ) and immunoblot analysis of uS3 and uS5 in sucrose gradient fractions from control hiPSCi, after short (2.5 min) or long (2 hours) treatment with 2 µg/ml homoharringtonine, or after treatment with 0.05 mg/l anisomycin (ANS) for 15 minutes ( n = 1 biological replicate). Membranes from Fig. were stripped and re-probed with antibodies against uS3 and uS5. i , Global protein synthesis measurements by OPP labeling in control hiPSCi and/or after treatment with 6.25, 12.5, and 25 µM 4EGI-1 for 1 day. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to the average value in controls ( n = 2 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Comparison, RNA Sequencing, Western Blot, Control, Labeling, Fluorescence, Flow Cytometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Schematic model of the consequences of ZNF598 depletion or ZNF598 RING expression in inducible hiPS cells. b , Polysome profiles (top) and immunoblot analysis of ZNF598 and uS5 in polysome gradient fractions (bottom) of inducible hiPS cells expressing sgControl, a ZNF598 sgRNA or ZNF598 RING ( n = 1 biological replicate). c , Metagene profiles of ribosomal A-site occupancy from monosome footprints around CDS start and stop sites ( n = 2 biological replicates). d , Volcano plot of differential ribosome pause sites upon ZNF598 RING expression in inducible hiPS cells (two-tailed Fisher’s exact test with Benjamini–Hochberg correction, adjusted P ≤ 0.01). e , Nucleotide (top) and amino acid (bottom) motif analysis of significantly increased pause sites in well-translated mRNAs (>0.5 footprints per codon in all samples; n = 3,421) in ZNF598 RING -expressing inducible hiPS cells. f , Volcano plot of differential ribosome pausing analysis upon ZNF598 knockdown (sgZNF598) in inducible hiPS cells as in d . g , Nucleotide (top) and amino acid (bottom) motif analysis of significantly increased pause sites in well-translated mRNAs (>0.5 footprints per codon in all samples; n = 2,463) in sgZNF598 inducible hiPS cells. h , i , GO term enrichment analysis of genes with significantly increased pause sites (one-tailed Fisher’s exact test with Benjamini–Hochberg correction, adjusted P ≤ 0.01) within the first five codons in ZNF598 RING -expressing inducible hiPS cells ( h ) and throughout the ORF in sgZNF598 inducible hiPS cells ( i ) filtered for TPM > 1 in RNA-seq from inducible hiPS cells. j , Distribution of monosome footprints (in reads per million (rpm)) along the H1-5 (left) and H3C2 (right) mRNA in control and ZNF598 RING -expressing inducible hiPS cells ( n = 2 biological replicates). Significant differential pauses are indicated with red arrows. k , Representative histograms of cell-cycle analysis in inducible hiPS cells by DNA staining with EdU followed by flow cytometry. l , Changes in the fraction of cells in different cell-cycle phases calculated by flow cytometry analysis after EdU staining ( n = 3 biological replicates; >10,000 cells per analysis; P values from an unpaired two-tailed t -test).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Western Blot, Two Tailed Test, Knockdown, One-tailed Test, RNA Sequencing, Control, Cell Cycle Assay, Staining, Flow Cytometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Venn diagram of overlaps between 5´ TOP motif mRNAs ( n = 97) and transcripts with significantly increased A-site pauses in the first 5 codons in ZNF598 RING -expressing hiPSCi ( n = 704), and comparison of 5´ UTR lengths and TPM levels from RNA-Seq in both groups ( p -value from two-sided Wilcoxon test). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. b , Comparison of ribosome recruitment scores for 5´ UTRs quantified by direct analysis of ribosome targeting (DART) for histone mRNAs with start site pauses ( n = 29), other mRNAs with start site pauses ( n = 351), other well-translated mRNAs ( n = 1340), and other detectable mRNAs ( n = 3791). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range. c , Density heatmaps of 50-80 nt ribosome footprints according to length and 5’ end position around CDS start (left) and stop sites (right) for a second biological replicate. d , Density heatmaps of 50-80 nt ribosome footprints according to length and 3’ end position around CDS start (left) and stop sites (right) for a second biological replicate. e , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in hiPSCi ( n = 2 biological replicates). f , Polysome profiles and immunoblot analysis of uS10 and eS10 in sucrose gradient fractions from control HEK293i or after a short (2.5 min) treatment with 2 µg/ml homoharringtonine (HAR) ( n = 1 biological replicate). g , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in ZNF598-depleted hiPSCi treated with GCN2 inhibitor (GCN2i, A-92, 1.25 µM ; n = 2 biological replicates). h , Polysome profiles (from Fig. ) and immunoblot analysis of uS3 and uS5 in sucrose gradient fractions from control hiPSCi, after short (2.5 min) or long (2 hours) treatment with 2 µg/ml homoharringtonine, or after treatment with 0.05 mg/l anisomycin (ANS) for 15 minutes ( n = 1 biological replicate). Membranes from Fig. were stripped and re-probed with antibodies against uS3 and uS5. i , Global protein synthesis measurements by OPP labeling in control hiPSCi and/or after treatment with 6.25, 12.5, and 25 µM 4EGI-1 for 1 day. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to the average value in controls ( n = 2 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Comparison, RNA Sequencing, Western Blot, Control, Labeling, Fluorescence, Flow Cytometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Growth assays of cells transduced with a sgRNA targeting EIF2S1 in HEK293i, hiPSCi, or NPC cells ( n = 2 biological replicates; line: average) and knockdown efficiency measurements of target genes compared to a non-targeting control (sgControl) by quantitative RT-PCR ( n = 2 biological replicates, each with n = 3 technical replicates). b , Venn diagrams of overlap between genes upregulated or downregulated in HEK293i or hiPSCi depleted for ZNF598 or HBS1L by CRISPRi ( p -values from one-tailed Fisher’s exact test). c,d , Gene ontology (GO) enrichment in differentially expressed mRNAs using the “Biological Process” function in ClusterProfiler (one-tailed Fisher’s exact test with Benjamini-Hochberg correction, P-adj ≤ 0.01) in knockdown hiPSCi ( b ) or HEK293i ( c ). e, f , Flow cytometry analysis of eIF2α phosphorylation ( d ) and p38 phosphorylation ( e ) upon depletion of ZNF598 or HBS1L in hiPSCi in comparison to cells transduced with a non-targeting sgRNA (sgControl; n = 3 biological replicates; matched samples). hiPSC treated with 2.5 µg tunicamycin (TM) for 2 hours to induce the ISR or with 0.05 mg/l anisomycin (ANS) for 15 minutes to induce the RSR served as a positive control. g , Growth assays of hiPSCi expressing a non-targeting sgRNA (sgControl, day 6) in the absence (-) or presence of inhibitors of GCN2 (GCN2i, A-92, 1.25 µM), PERK (PERKi, GSK2606414, 4 nM), p38 (p38i, SB203580, 1 µM), or the ISR (ISRIB, 50 nM) in comparison to uninduced controls ( n = 3 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Transduction, Knockdown, Control, Quantitative RT-PCR, One-tailed Test, Flow Cytometry, Phospho-proteomics, Comparison, Positive Control, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Global protein synthesis measurements in knockdown inducible hiPS cells or inducible HEK293 cells by HPG labeling for 30 min. Median fluorescence intensity was quantified by flow cytometry (>10,000 cells per analysis) and normalized to values from cells transduced with sgControl ( n = 5 biological replicates; P values from an unpaired two-tailed t -test). b , LDH measurements in culture supernatants from knockdown inducible hiPS cells or inducible HEK293 cells. Values were normalized to supernatant from cells transduced with sgControl ( n = 4 technical replicates and 4 biological replicates; P values from an unpaired two-sided t -test). c , Heatmap of mRNAs differentially expressed upon ZNF598 or HBS1L repression in inducible hiPS cells or inducible HEK293 cells ( n = 2 biological replicates; two-sided Wald test with Benjamini–Hochberg correction, adjusted P ≤ 0.05; n = 3165). d , Heatmap of a data subset from c showing genes upregulated downstream of ATF4 within the ISR or downstream of ATF6 or IRE1α upon ER protein folding perturbations . e , Growth assays of inducible hiPS cells expressing an sgRNA targeting ZNF598 (day 6) or HBS1L (day 8) in the absence (−) or presence of GCN2i (A-92, 1.25 µM), PERKi (GSK2606414, 4 nM), p38i (SB203580, 1 µM) or ISRIB (50 nM) in comparison to uninduced controls ( n = 3 biological replicates; P values from an unpaired two-tailed t -test). f , Growth assays of inducible hiPS cells expressing an sgRNA targeting ZNF598 , HBS1L , GCN2 and PERK alone or in combination in comparison to uninduced controls ( n = 5 biological replicates; P values from an unpaired two-tailed t -test). g , Immunoblot analysis of eIF2α phosphorylation, p38 phosphorylation and total eIF2α and p38 levels in inducible hiPS cells and inducible HEK293 cells ( n = 4 biological replicates). Inducible hiPS cells treated with 2.5 µM tunicamycin (TM) for 2 h served as a positive control for induction of eIF2α phosphorylation; inducible hiPS cells treated with 0.05 mg L −1 ANS for 15 min served as a positive control for induction of p38 phosphorylation. Rep, replicate. h , Quantification of signal intensity in g by densitometry ( P values from an unpaired two-tailed t -test).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Knockdown, Labeling, Fluorescence, Flow Cytometry, Transduction, Two Tailed Test, Expressing, Comparison, Western Blot, Phospho-proteomics, Positive Control

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Immunoblot analysis of wild-type hiPSCi and hiPSC transduced with cDNA encoding HA-tagged wild-type (ZNF598 WT ) or C29S/C32S (ZNF598 RING ) ZNF598 ( n = 2 biological replicates). b , Stalling readthrough of reporters containing an AAA-encoded stretch of twenty lysines (AAA 20 ) in control hiPSCi and hiPSCi transduced with ZNF598 WT or ZNF598 RING cDNA. The median fluorescence intensity for BFP and mOrange was quantified by flow cytometry ( > 20,000 cells/ analysis) and normalized to average values for the hiPSCi control ( n = 3 biological replicates; P -values from unpaired two-tailed t-test). c , Global protein synthesis measurements by OP-Puromycin and AF647 detection. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to average values for the control ( n = 6 biological replicates; p -values from unpaired two-tailed t-test). d , Lactate dehydrogenase (LDH) measurements in culture supernatants from wild type or ZNF598 RING -expressing hiPSCi two days after transduction. Values were normalized to supernatant from wild type hiPSCi ( n = 4 technical replicates and 5 biological replicates; P -values from unpaired two-tailed t-test). e,f , Differential gene expression analysis in hiPSCi upon ZNF598 knockdown (sgZNF598) in comparison to a non-targeting sgRNA (sgControl), or in ZNF598 RING -expressing hiPSCi in comparison to untransduced hiPSCi ( n = 2 biological replicates). ( e ) Heatmap of mRNAs differentially expressed in at least one context (two-sided Wald test; P-adj ≤ 0.05; n = 2289). ( f ) Heatmap of a subset from ( e ) showing genes associated with the ISR. g , Flow cytometry analysis of eIF2α (top) or p38 (bottom) phosphorylation upon ZNF598 RING expression in hiPSCi in comparison to cells transduced with a non-targeting sgRNA (sgControl; n = 2 biological replicates) on day 1-3 after transduction. h , Gene ontology (GO) enrichment in differentially expressed mRNAs from ( e ) using the “Biological Process” function in ClusterProfiler (one-tailed Fisher’s exact test with Benjamini-Hochberg correction, P-adj ≤ 0.01).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Western Blot, Transduction, Control, Fluorescence, Flow Cytometry, Two Tailed Test, Expressing, Gene Expression, Knockdown, Comparison, Phospho-proteomics, One-tailed Test

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Growth assays of cells transduced with a sgRNA targeting EIF2S1 in HEK293i, hiPSCi, or NPC cells ( n = 2 biological replicates; line: average) and knockdown efficiency measurements of target genes compared to a non-targeting control (sgControl) by quantitative RT-PCR ( n = 2 biological replicates, each with n = 3 technical replicates). b , Venn diagrams of overlap between genes upregulated or downregulated in HEK293i or hiPSCi depleted for ZNF598 or HBS1L by CRISPRi ( p -values from one-tailed Fisher’s exact test). c,d , Gene ontology (GO) enrichment in differentially expressed mRNAs using the “Biological Process” function in ClusterProfiler (one-tailed Fisher’s exact test with Benjamini-Hochberg correction, P-adj ≤ 0.01) in knockdown hiPSCi ( b ) or HEK293i ( c ). e, f , Flow cytometry analysis of eIF2α phosphorylation ( d ) and p38 phosphorylation ( e ) upon depletion of ZNF598 or HBS1L in hiPSCi in comparison to cells transduced with a non-targeting sgRNA (sgControl; n = 3 biological replicates; matched samples). hiPSC treated with 2.5 µg tunicamycin (TM) for 2 hours to induce the ISR or with 0.05 mg/l anisomycin (ANS) for 15 minutes to induce the RSR served as a positive control. g , Growth assays of hiPSCi expressing a non-targeting sgRNA (sgControl, day 6) in the absence (-) or presence of inhibitors of GCN2 (GCN2i, A-92, 1.25 µM), PERK (PERKi, GSK2606414, 4 nM), p38 (p38i, SB203580, 1 µM), or the ISR (ISRIB, 50 nM) in comparison to uninduced controls ( n = 3 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Transduction, Knockdown, Control, Quantitative RT-PCR, One-tailed Test, Flow Cytometry, Phospho-proteomics, Comparison, Positive Control, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Global protein synthesis measurements in knockdown inducible hiPS cells or inducible HEK293 cells by HPG labeling for 30 min. Median fluorescence intensity was quantified by flow cytometry (>10,000 cells per analysis) and normalized to values from cells transduced with sgControl ( n = 5 biological replicates; P values from an unpaired two-tailed t -test). b , LDH measurements in culture supernatants from knockdown inducible hiPS cells or inducible HEK293 cells. Values were normalized to supernatant from cells transduced with sgControl ( n = 4 technical replicates and 4 biological replicates; P values from an unpaired two-sided t -test). c , Heatmap of mRNAs differentially expressed upon ZNF598 or HBS1L repression in inducible hiPS cells or inducible HEK293 cells ( n = 2 biological replicates; two-sided Wald test with Benjamini–Hochberg correction, adjusted P ≤ 0.05; n = 3165). d , Heatmap of a data subset from c showing genes upregulated downstream of ATF4 within the ISR or downstream of ATF6 or IRE1α upon ER protein folding perturbations . e , Growth assays of inducible hiPS cells expressing an sgRNA targeting ZNF598 (day 6) or HBS1L (day 8) in the absence (−) or presence of GCN2i (A-92, 1.25 µM), PERKi (GSK2606414, 4 nM), p38i (SB203580, 1 µM) or ISRIB (50 nM) in comparison to uninduced controls ( n = 3 biological replicates; P values from an unpaired two-tailed t -test). f , Growth assays of inducible hiPS cells expressing an sgRNA targeting ZNF598 , HBS1L , GCN2 and PERK alone or in combination in comparison to uninduced controls ( n = 5 biological replicates; P values from an unpaired two-tailed t -test). g , Immunoblot analysis of eIF2α phosphorylation, p38 phosphorylation and total eIF2α and p38 levels in inducible hiPS cells and inducible HEK293 cells ( n = 4 biological replicates). Inducible hiPS cells treated with 2.5 µM tunicamycin (TM) for 2 h served as a positive control for induction of eIF2α phosphorylation; inducible hiPS cells treated with 0.05 mg L −1 ANS for 15 min served as a positive control for induction of p38 phosphorylation. Rep, replicate. h , Quantification of signal intensity in g by densitometry ( P values from an unpaired two-tailed t -test).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Knockdown, Labeling, Fluorescence, Flow Cytometry, Transduction, Two Tailed Test, Expressing, Comparison, Western Blot, Phospho-proteomics, Positive Control

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Immunoblot analysis of wild-type hiPSCi and hiPSC transduced with cDNA encoding HA-tagged wild-type (ZNF598 WT ) or C29S/C32S (ZNF598 RING ) ZNF598 ( n = 2 biological replicates). b , Stalling readthrough of reporters containing an AAA-encoded stretch of twenty lysines (AAA 20 ) in control hiPSCi and hiPSCi transduced with ZNF598 WT or ZNF598 RING cDNA. The median fluorescence intensity for BFP and mOrange was quantified by flow cytometry ( > 20,000 cells/ analysis) and normalized to average values for the hiPSCi control ( n = 3 biological replicates; P -values from unpaired two-tailed t-test). c , Global protein synthesis measurements by OP-Puromycin and AF647 detection. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to average values for the control ( n = 6 biological replicates; p -values from unpaired two-tailed t-test). d , Lactate dehydrogenase (LDH) measurements in culture supernatants from wild type or ZNF598 RING -expressing hiPSCi two days after transduction. Values were normalized to supernatant from wild type hiPSCi ( n = 4 technical replicates and 5 biological replicates; P -values from unpaired two-tailed t-test). e,f , Differential gene expression analysis in hiPSCi upon ZNF598 knockdown (sgZNF598) in comparison to a non-targeting sgRNA (sgControl), or in ZNF598 RING -expressing hiPSCi in comparison to untransduced hiPSCi ( n = 2 biological replicates). ( e ) Heatmap of mRNAs differentially expressed in at least one context (two-sided Wald test; P-adj ≤ 0.05; n = 2289). ( f ) Heatmap of a subset from ( e ) showing genes associated with the ISR. g , Flow cytometry analysis of eIF2α (top) or p38 (bottom) phosphorylation upon ZNF598 RING expression in hiPSCi in comparison to cells transduced with a non-targeting sgRNA (sgControl; n = 2 biological replicates) on day 1-3 after transduction. h , Gene ontology (GO) enrichment in differentially expressed mRNAs from ( e ) using the “Biological Process” function in ClusterProfiler (one-tailed Fisher’s exact test with Benjamini-Hochberg correction, P-adj ≤ 0.01).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Western Blot, Transduction, Control, Fluorescence, Flow Cytometry, Two Tailed Test, Expressing, Gene Expression, Knockdown, Comparison, Phospho-proteomics, One-tailed Test