Fig. 5.

Pum1 and Pum2 autoregulate each other and bind overlapping sets of functionally related mRNAs in ESCs. (A) Pum1 and Pum2 participate in an interregulatory loop (Upper); Pum1 and Pum2 mRNA have multiple putative PRE sites in their 3′ UTR (Middle: red lines, strict canonical TGTANATA motif; black lines, degenerate TDTANAWH motif). Western blotting analysis reveals increased Pum2 levels in Pum1^−/− ESCs (Lower Left) and increased Pum1 levels in Pum2^−/− ESCs (Lower Right). (B) Scatter plot of the log intensity of IP samples (y axis) and of negative controls (incubated with blocking peptide against which the antibody was raised [x axis]). (C) Heat map of enrichment in IP and control samples for the top 47 gene probes. (D) qRT-PCR of known Pum1 mRNA targets confirming enrichment in IP samples; error bars indicate SEM of three replicates. (E) GSEA of top 500 Pum1 mRNA targets by fold enrichment and P value. (F) Venn diagram indicating total number of significantly enriched (P < 0.001, fold enrichment >1.5) mRNA targets of Pum1 (pink) and Pum2 (green). (G) GSEA of the 354 overlapping mRNA targets of Pum1 and Pum2.