Fig. 1: P-body-seq permits comprehensive profiling of P-body contents.
From: Selective RNA sequestration in biomolecular condensates directs cell fate transitions
a, Schematic for the purification and transcriptomic profiling of P-bodies from HEK293T cells based on GFP-LSM14A expression. b, Representative IF imaging of GFP-LSM14A puncta (green), colocalizing with EDC4 puncta (red) in HEK293T cells. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue) (scale bar, 10 μm). n = 3 independent experiments. c, Representative flow cytometry plots showing gating for GFP-LSM14A+ P-bodies in HEK293T cells. SSC, side scatter. d, Representative imaging of GFP-LSM14A puncta (green) in control and DDX6-KO HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale bar, 10 μm) (left). P-body number in control (n = 50 cells) and DDX6-KO (n = 50 cells) HEK293T cells (right). Unpaired two-sided Student’s t-test, median ± 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 biological independent samples per group, P < 0.05). FC, fold change; FPKM, fragments per kilobase of transcript per million mapped reads. g, GO pathway analysis of P-body-enriched mRNA in HEK293T cells, using expressed genes as background. Enrichment was tested using two-sided Fisher’s exact test with multiple-testing correction (Benjamini–Hochbergfalse discovery rate (FDR)). Padj, adjusted P value. h, GO pathway analysis of cytoplasm-enriched mRNA in HEK293T cells, using expressed genes as background. Enrichment was tested using two-sided Fisher’s exact test with multiple-testing correction (Benjamini–Hochberg FDR). i, Representative FISH imaging of POLK RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green). Nuclei were counterstained with DAPI (blue) (scale bar, 10 μm). n = 3 independent experiments. j, Quantification of POLK mRNA molecules in P-bodies based on FISH (n = 50 cells; right) and P-body sequencing (right); median ± s.d. RPKM, reads per kilobase per million mapped reads. k, Read coverage distribution over the gene body of the longest annotated isoforms for genes enriched in P-bodies or the cytoplasm (cyto) in HEK293T cells. l, PolyA tail length as determined in ref. 50 compared to P-body enrichment based on Smart-seq and snapTotal-seq (Pearson correlation test). m, Translation efficiency (TE; log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in HEK293T cells (Pearson correlation test (two sided), P = 3.94 × 10−98).
