solid sequences free of gaps or stop codons  (CodonCode corporation)

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) Schematic for the purification and transcriptomic profiling of P-bodies from HEK293T cells based on the expression of GFP-LSM14A. (b) Representative IF imaging of GFP-LSM14A puncta (green), colocalizing with EDC4 puncta (red) in HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale: 10mm). (c) Representative flow cytometry plots showing gating for GFP-LSM14A+ P-bodies in HEK293T cells. (d) Representative imaging of GFP-LSM14A puncta (green) in control and DDX6 KO HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale: 10mm) (left panel). P-body number in control (n=50 cells) and DDX6 KO (n=50 cells) HEK293T cells (right panel). Unpaired Student’s t-test, mean ± s.d., ****: p<0.0001. (e) Representative flow cytometry plots showing gating for GFP-LSM14A+ P-bodies in control and DDX6 KO HEK293T cells. (f) MA plot of RNA-seq data depicting P-body enriched genes in red and cytoplasm enriched genes in blue in HEK293T cells (n=2, p < 0.05). (g) GO pathway analysis of P-body enriched mRNAs in HEK293T cells. (h) GO pathway analysis of cytoplasmic fraction-enriched mRNAs in HEK293T cells. (i) Representative FISH imaging of POLK RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green). Nuclei were counterstained with DAPI (blue) (scale: 10mm). (j) Quantification of POLK mRNA molecules in P-bodies based on FISH (n=50 cells; right) and P-body sequencing (right). (k) Read coverage distribution over the gene body of the longest annotated isoforms for genes enriched in P-bodies or cytoplasm in HEK293T cells. (l) PolyA tail length as determined in4 compared to P-body enrichment based on SMART-Seq and SnapTotal-Seq, Pearson correlation test. (m) Translation efficiency (log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in HEK293T cells, Pearson correlation test.

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: Purification, Expressing, Imaging, Flow Cytometry, Control, RNA Sequencing, Sequencing

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) Representative image of pre-sorted cell lysate containing GFP-LSM14A + P-bodies (scale: 10μm). (b) Representative imaging of GFP-LSM14A puncta (green) in control and DDX6 KD HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale: 10μm) (left panel). P-body number in control (n=50 cells) and DDX6 KD (n=50 cells) HEK293T cells (right panel). Unpaired Student’s t-test, mean ± s.d., ****: p<0.0001. (c) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in control and DDX6 KD HEK293T cells. (d) Representative flow cytometry plots showing gating for SYTOX BLUE + events in GFP + and GFP - gates of pre-sorted cytoplasmic fraction from HEK293T cells. (e) Venn diagram showing the overlap between P-body-associated mRNAs in HEK293T from SMART-Seq and SnapTotal-Seq. (f) Venn diagram showing the overlap between P-body-associated mRNAs in HEK293T of this study (blue) and a published dataset from (grey). (g) Length (left panel) and GC content (right panel) density plots of mRNAs in purified P-body and cytoplasmic fractions of HEK293T cells.

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: Imaging, Control, Flow Cytometry, Purification

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) Normalized Enrichment Score (NES) of primed human ES cell-related genes in purified P-bodies from endoderm and mesoderm progenitors. (b) A schematic of the strategy for P-body dissolution in human endoderm progenitors during their differentiation to hepatocytes. (c) Representative IF imaging of AFP (green) positive cells in hPSCs upon DDX6 KO compared to control cells. Nuclei were counterstained with DAPI (blue) (scale: 100μm) (Left panel). Quantification of AFP + cells upon DDX6 suppression. Unpaired Student’s t-test, sgControl (n=3 fields), sgDDX6 (n=3 fields), mean ± s.d., ****: p<0.0001 (Right panel). (d) qRT-PCR analysis for the indicated genes in hepatocytes. Unpaired Student’s t-test, mean ± s.d. ( n=3 ), (**p<0.01, ****p<0.0001). (e) A schematic of neuron maturation. (f) Venn diagram showing the overlap between P-body-associated mRNAs in neurons cultured for 7 and 20 days. (g) Normalized Enrichment Score (NES) of neural progenitor-related genes in neurons cultured for 7 and 20 days. (h) A schematic of endoderm progenitor maturation. (i) Normalized Enrichment Score (NES) of primed human ES cell-related genes in endoderm and mature endoderm progenitors. (j) GSEA analysis showing enrichment for a naïve signature in purified P-bodies from primed ES cells. (k) GSEA analysis showing enrichment for an 8C-like signature in purified P-bodies from naïve ES cells.

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: Purification, Dissolution, Imaging, Control, Quantitative RT-PCR, Cell Culture

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) Schematic of the conversion from naïve to primed state mouse ES cells (left panel). Representative bright field images of naïve and primed mouse ES cells (scale: 50μm) (right panel). (b) Heatmap showing expression levels of naïve-specific and primed-specific transcripts. (n=2). (c) Representative IF imaging of EDC4 puncta (green) in naïve and primed mouse ES cells. Cell membranes were labeled with Phalloidin (red) and nuclei were counterstained with DAPI (blue) (scale: 10μm) (left panel). P-body number in naïve (n=60 cells) and primed (n=60 cells) mouse ES cells (right panel). Unpaired Student’s t-test, mean ± s.d., n.s.: p >0.05. (d) Representative flow cytometry plots showing gating for GFP-LSM14A+ P-bodies in naïve and primed mouse ES cells. (e) Heatmap showing expression levels of differentially enriched mRNAs between purified P-body fractions of naïve and primed mouse ES cells, with GO pathway analysis of P-body-enriched transcripts for the indicated clusters. Gene number in each transcript cluster is indicated in the figure (n=2, p < 0.05). (f) GSEA analysis of blastomere-related genes in purified P-body fraction vs. cytoplasmic fraction from naïve mouse ES cells, NES=1.28, p=0.006. (g) A schematic of the strategy for P-body dissolution in mouse naïve ES cells carrying blastomere-specific reporters (MERVL-tdTomato and ZSCAN4-GFP). (h, i) Flow cytometric quantification of MERVL-tdTomato+ and ZSCAN4-GFP+ cells upon Lsm14a , Ddx6, and Eif4enif1 KO in mouse naïve ES cells. Control (n=3), Lsm14a (n=3), Ddx6 KO (n=3) and Eif4enif1 KO (n=3).

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: Expressing, Imaging, Labeling, Flow Cytometry, Purification, Dissolution, Control

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

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) Representative western blot showing DDX6 protein levels in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. (b) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. (c) Representative IF imaging of EDC4 puncta (red) in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, treated with dTAG-13 for 3 and 9 hours. Nuclei were counterstained with DAPI (blue) (scale: 10mm). (d) GO terms for P-body enriched genes with increased ribosome occupancy of Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells following dTAG13 treatment for 6 hours). (e) Volcano plot of RNA-seq data depicting differential expression of total RNA fraction in Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells following dTAG13 treatment for 6 hours, with P-body enriched genes in red and cytoplasm enriched genes in blue (n=3, p < 0.05). (f) GO terms of P-body enriched genes that showed downregulated gene expression in Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells following dTAG13 treatment for 6 hours.

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: Western Blot, Flow Cytometry, Imaging, RNA Sequencing, Quantitative Proteomics, Gene Expression

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) A schematic of the strategy for miR-300 inhibition in mouse naïve ES cells. (b) qRT-PCR analysis of the expression for miR-300 targets in P-bodies of mouse naïve ES cells after miR-300 inhibition and for control. n=3, unpaired Student’s t-test, mean ± s.d., *p<0.05, **p<0.01, ****p<0.0001. (c) Heatmap showing expression levels of differentially enriched 2C-related genes in mouse naïve ES cells after miR-300 inhibition and for control. (d) A schematic of the strategy for the generation of Let7 wt and Let7 mut reporter cell lines. (e) Representative IF imaging of EDC4 puncta (green), and Nanog-MS2 (red) in Nanog Let7 wt (upper panel) and Nanog Let7 mut cells (lower panel). Nuclei were counterstained with DAPI (blue) (scale: 10μm). (f) qRT-PCR analysis of Nanog expression in Nanog Let7 wt and Nanog Let7 mut cells compared to CTRL cells (Nanog KO). (g) Representative western blot showing NANOG protein levels in Nanog Let7 wt and Nanog Let7 mut cells compared to CTRL cells ( Nanog KO). (h-j) Representative pictures (h) and quantification of Alkaline Phosphatase staining of cell colony number (i) and size (j) from Nanog Let7 wt and Nanog Let7 mut cells cultured in FBS+LIF conditions. (k) Heatmap showing expression levels of differentially enriched 8C-related mRNAs between purified P-body and cytoplasmic fractions in human naïve ES cells. (n=2). (l) A schematic of the strategy for P-body dissolution in naïve human ES cells carrying blastomere-specific reporter (TPRX1-GFP). (m) Quantification of TPRX1-GFP + cells upon DDX6 KD. Unpaired Student’s t-test, control (n=3), DD X6 KD (n=3), mean ± s.d., ****: p<0.0001. (n) Representative IF imaging of H3Y1 (green) positive cells in naïve human ES cells upon DDX6 KO compared to control cells. Nuclei were counterstained with DAPI (blue) (scale: 50μm) (Left panel). Quantification of H3Y1 + cells upon DDX6 suppression. Unpaired Student’s t-test, sgControl (n=5 fields), sgDDX6 (n=8 fields), mean ± s.d., ****: p<0.0001 (Right panel). (o) GSEA analysis for hPGCLC-related gene expression signature in purified P-bodies from human primed ES cells. (p) A schematic of hiPSC to PGCLC differentiation. HiMeLCs: human incipient mesoderm-like cells. (q) Flow cytometric analysis of TFAP2C-GFP and BLIMP1-TOMATO expression after four days of PGCLC differentiation in 3D aggregates (left). Quantification of TFAP2C-GFP + /BLIMP1-TOMATO + cells by flow cytometry for three experiments (right). Error bars indicate mean ± s.d. ( n=3 ), statistical significance was determined using a two-tailed unpaired Student’s t-test (***P<0.001). (r) Quantitative RT-PCR analysis for the indicated genes after four days of PGCLC differentiation in 3D aggregates. Error bars indicate mean ± s.d. ( n>3 ), statistical significance was determined using a two-tailed unpaired Student’s t-test (****P<0.0001).

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: Inhibition, Quantitative RT-PCR, Expressing, Control, Imaging, Western Blot, Staining, Cell Culture, Purification, Dissolution, Gene Expression, Flow Cytometry, Two Tailed Test