Journal: bioRxiv
Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions
doi: 10.1101/2025.05.08.652299
Figure Lengend Snippet: (a) Translation efficiency (log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in naïve and primed mouse ES cells. Ribosome profiling data from , Pearson correlation test. (b) A schematic showing CRISPR-Cas9-based homozygous insertion of FKBP12F36V-HA-P2A-mCherry sequence in place of the stop codon of the endogenous Ddx6 allele. (c) Representative intracellular flow cytometry plots for DDX6 in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. (d) Representative IF imaging of EDC4 puncta (red) in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. Nuclei were counterstained with DAPI (blue) (scale: 10μm). (e) P-body number in Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. DMSO (n=70 cells), 3 hour-dTAG13 (n=70 cells), 6 hour-dTAG13 (n=70 cells), 9 hour-dTAG13 (n=70 cells), unpaired Student’s t-test, mean ± s.d., ****: p <0.0001. (f) Cumulative distribution function (CDF) plot showing ribosome occupancy (log2 FC) of P-body enriched and P-body-depleted mRNAs for untreated (DMSO) vs. dTAG-13 treated (6hrs) Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells, ks-test. (g) Box plots showing the change in ribosome occupancy of all P-body enriched genes compared to all other genes. Unpaired t-test, mean ± s.d, padj (Holm’s method). (h) Normalized Enrichment Score (NES) of gene sets from (2C) , (Naïve) , and (Primed) , p<0.05. (i) Box plots showing the change in ribosome occupancy of P-body enriched 2C-related genes compared to non-P-body enriched 2C genes. Unpaired t-test, mean ± s.d, padj (Holm’s method). (j) Box plots showing the change in protein levels of all P-body enriched genes compared to P-body depleted genes, after 1 day and 3 days of dTAG-13 treatment. Unpaired t-test, mean ± s.d, padj (Holm’s method) (* p < 0.05, ** p < 0.01, **** p < 0.0001) (k) Box plots showing the change in protein levels of P-body enriched 2C-related genes compared to P-body depleted genes, after 1 day and 3 days of dTAG-13 treatment. Unpaired t-test, mean ± s.d, padj (Holm’s method). (l) Heatmap showing protein levels of 2C-related genes after 1 day and 3 days of dTAG-13 treatment compared to control samples.
Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).
Techniques: RNA Sequencing, CRISPR, Sequencing, Flow Cytometry, Imaging, Control
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