NRDE2 is required for the proper recruitment of CCDC174 to 5′SSs. (A) Dual-luciferase assay of the transiently transfected CDK2 and TTI1 reporters in cells allowing simultaneous depletion of NRDE2 and CCDC174. Plotted is the average value of normalized Renilla luciferase activity from three independent experiments each performed in triplicate transfections. Error bars represent standard deviation. (B) Formaldehyde cross-linking TAP-MS analysis of 3A-CCDC174 compared with untagged control and performed in three independent replicates for each sample. Same labeling scheme as in Figure 1, A and B, applies. (C) Live-cell spinning-disk microscopy of endogenous mNeonGreen-tagged NRDE2 and CCDC174 with endogenous mCherry-tagged U2AF2 in cells allowing depletion of CCDC174 and NRDE2, respectively. Scale bar 10 µm. (D) Quantification of live-cell spinning-disk microscopy fluorescence signal intensity for endogenously tagged mNeonGreen-NRDE2 in WT and CCDC174-depleted cells, and mNeonGreen-CCDC174 in WT and Nrde2-KO cells. Each dot represents a single nucleus. Statistical significance was assessed by unpaired two-tailed t-test. (E) Heat maps of the normalized CCDC174 BCLIP-seq signal intensity in WT and Nrde2-KO cells centered at annotated 5′SSs of all transcripts expressed in mESCs. Splice sites are ordered by decreasing CCDC174 signal in the WT sample. (F) Metaplots of normalized CCDC174 BCLIP-seq signal around all annotated expressed 5′SSs in WT (solid line; same data as in Fig. 2D) and Nrde2-KO (dashed line).
