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