NRDE2 regulates the splicing of introns with weak 5′SSs. (A) Differential gene expression based on duplicate RNA-seq analysis of Nrde2-KO (4 d of 0.1 µM 4OHT treatment), Nrde2Δ200, Nrde2-D174R, Ccdc174-dTAG (24 h of 0.5 µM dTAG-13 treatment), and Mtrex-KO (3 d of 0.1 µM 4OHT treatment) cells compared with the matching untreated and wild-type controls (WT). Heat map consists of genes differentially expressed (fold change >2, adjusted P-value <0.01) in Nrde2-KO cells. Genes are divided into four clusters depending on expression changes in Nrde2-KO and Mtrex-KO samples. (B) Scatter plot comparing differential gene expression in Nrde2-KO and Mtrex-KO cells relative to the corresponding WT cells. Each dot represents a single gene. Genes up-regulated in 2C-like cells (Macfarlan et al. 2012) are highlighted in red. (C) Metaplots of normalized NRDE2, CCDC174, and EIF4A3 BCLIP-seq signal around 5′SSs of introns up-regulated (solid line) or not up-regulated (dashed line) in Nrde2-KO cells compared with WT. (D) Overlap of NRDE2 target introns (introns with a NRDE2 BCLIP-seq peak at their 5′SS) up-regulated in Nrde2-KO, Ccdc174-dTAG, or Mtrex-KO cells. Note that the total number of introns up-regulated might differ depending on whether protein depletion (Ccdc174-dTAG) or conditional gene knockout strategies were used (Nrde2-KO, Mtrex-KO). (E) Density plots of the distribution of NRDE2 target introns up-regulated (blue line) or not up-regulated (gray line) in Nrde2-KO cells based on their 5′SS, 3′SS, and branch point strength. P-values were calculated using the Wilcoxon rank-sum test. (F) Differential expression (RNA-seq) of genes containing NRDE2 target introns in Nrde2-KO cells (4 d of 0.1 µM 4OHT treatment) untreated (left) or treated (right) for 4 h with 100 µg/mL CHX. Genes are split into two groups: those containing only target introns not up-regulated (gray) and those containing at least one target intron up-regulated in Nrde2-KO cells untreated (blue) or treated with CHX (purple). Bottom boxplots show differential ribosome occupancy for the same genes in Nrde2-KO cells relative to WT. P-values were calculated using the Wilcoxon rank-sum test. (G) UCSC genome browser screenshot showing normalized BCLIP-seq, RNA-seq, and Ribo-seq tracks over Dnmt1 gene locus. Position of the NRDE2 target intron 10 is highlighted in red.
