Figure 7. Arid5a binds rRNAs and... | Rockefeller University Press

$Arid5a binds rRNAs and facilitates protein synthesis in murine stromal cells. (a) Experimental design. Inset: ST2 cells were treated with murine IL-17 for 1, 3, or 6 h, and Arid5a and β-actin were assessed by immunoblotting, representative of two independent experiments. (b) ST2 cells were treated ± IL-17 for 3 h and lysates were subjected to RIP with anti-Arid5a Abs or IgG controls. Enrichment of rRNAs corresponding to mouse 18S, 5S, 5.8S, and 28S were determined by qPCR, normalized to untreated IgG RIP controls. Data show mean ± SEM, analyzed by one-way ANOVA with the Kruskal–Wallis test. Each symbol represents an individual experimental sample (18S n = 10; 5S, 5.8S, and 28S n = 6), pooled from two independent experiments. (c) ST2 cells were treated ± IL-17 for 3 h and lysates were subjected to fRIP-Seq by Illumina with anti-Arid5a Abs. The plot shows differentially expressed mRNAs in IL-17–treated pulldowns compared to untreated controls (n = 3–4 independent replicates). NS, not significant. (d) GSEA pathway predictions of Arid5a-fRIP-Seq, comparing untreated controls and IL-17–treated samples. FDR < 0.25, minimum gene IDs in category = 15. (e) Intersection analysis comparing orthologs in Arid5a RIP-Seq from IL-17–treated ST2 and HK-2 cells (from ). Selected genes identified in the intersection dataset. (f) ST2 cells were treated ± IL-17 for 3 h. Cytoplasmic lysates were separated by sucrose gradient fractionation. Arid5a and RPL7A were assessed in two pooled fractions per sample. Data are representative of two independent experiments. (g) ST2 cells were treated ± IL-17 for 3 h, stained with DAPI or Arid5a and RPL7A, and imaged by confocal microscopy. Arrows denote regions of co-association of Arid5a with RPL7A. Size bar = 10 µm. Quantitation of nuclear Arid5a is shown on the right. Each value represents the percentage of colocalized volume from an individual cell (n = 17–31). Analyzed by Student’s t test. *P < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Source data are available for this figure: SourceData F7.$

Figure 7.

Arid5a binds rRNAs and facilitates protein synthesis in murine stromal cells. (a) Experimental design. Inset: ST2 cells were treated with murine IL-17 for 1, 3, or 6 h, and Arid5a and β-actin were assessed by immunoblotting, representative of two independent experiments. (b) ST2 cells were treated ± IL-17 for 3 h and lysates were subjected to RIP with anti-Arid5a Abs or IgG controls. Enrichment of rRNAs corresponding to mouse 18S, 5S, 5.8S, and 28S were determined by qPCR, normalized to untreated IgG RIP controls. Data show mean ± SEM, analyzed by one-way ANOVA with the Kruskal–Wallis test. Each symbol represents an individual experimental sample (18S n = 10; 5S, 5.8S, and 28S n = 6), pooled from two independent experiments. (c) ST2 cells were treated ± IL-17 for 3 h and lysates were subjected to fRIP-Seq by Illumina with anti-Arid5a Abs. The plot shows differentially expressed mRNAs in IL-17–treated pulldowns compared to untreated controls (n = 3–4 independent replicates). NS, not significant. (d) GSEA pathway predictions of Arid5a-fRIP-Seq, comparing untreated controls and IL-17–treated samples. FDR < 0.25, minimum gene IDs in category = 15. (e) Intersection analysis comparing orthologs in Arid5a RIP-Seq from IL-17–treated ST2 and HK-2 cells (from ). Selected genes identified in the intersection dataset. (f) ST2 cells were treated ± IL-17 for 3 h. Cytoplasmic lysates were separated by sucrose gradient fractionation. Arid5a and RPL7A were assessed in two pooled fractions per sample. Data are representative of two independent experiments. (g) ST2 cells were treated ± IL-17 for 3 h, stained with DAPI or Arid5a and RPL7A, and imaged by confocal microscopy. Arrows denote regions of co-association of Arid5a with RPL7A. Size bar = 10 µm. Quantitation of nuclear Arid5a is shown on the right. Each value represents the percentage of colocalized volume from an individual cell (n = 17–31). Analyzed by Student’s t test. *P < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Source data are available for this figure: SourceData F7.

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