anti rps2 leukemia  (Bethyl)

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