Fig. 6: Dynamic RNA binding of G3BP1 during the assembly of SGs.
From: Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing
a, Immunofluorescence imaging showing the localization of G3BP1 in HeLa cells without treatment (T0) and with 0.5 mM NaAsO2 treatment for 10 min (T10), 20 min (T20) and 60 min (T60), respectively. Scale bars, 5 μm. b, Immunofluorescence imaging (top) showing that G3BP1 (yellow) was colocalized with biotinylated cDNA (green) generated from ARTR-seq. The line graph analysis (bottom) shows the relative fluorescence intensity along the line. Scale bar, 5 μm. c, A Venn diagram showing the overlap between the G3BP1–RNA targets at T0 and T60. d, A box plot exhibiting SG enrichment of RNA targets from three groups defined in c, including T0 only (T0_only, n = 965) fraction, T0 and T60 overlapped (OL, n = 274) fraction and T60 only (T60_only, n = 482) fraction. SG enrichment values were reported in SG RNA-seq46. P values were determined by the two-tailed Wilcoxon test. e, KEGG enrichment analysis showing RNA targets from three groups are enriched in distinct pathways. P values were calculated by the clusterProfiler package53 using the one-tailed hypergeometric test. f,g, Boxplots of G3BP1 binding strength for SG-enriched RNAs (n = 1,512, f) and SG-depleted RNAs (n = 1,671, g). G3BP1 binding strength was defined as ARTR-seq reads log2FC(G3BP1/input). SG-enriched RNAs and SG-depleted RNAs were obtained from a previous SG RNA-seq report46. h, A heatmap (left) depicting changing patterns of G3BP1 binding strength for RNA clusters across time. RNAs were ranked from large to small according to the s.d. of G3BP1 binding intensity over different time intervals, and the top 50% of RNAs were selected and clustered by fuzzy c-means. Line plots (right) exhibit the corresponding change of G3BP1 binding strength in each cluster. Each line represents one gene, with the black line being the centroid of the cluster. i, IGV snapshots showing two G3BP1–RNA targets with decreased (left) and increased (right) binding strength, and each panel was normalized by counts per million. Heatmaps (bottom) show G3BP1 binding strength with the size of circles representing its absolute value.
