NRDE2 and CCDC174 bind to U1 snRNA. (A) Proportion of BCLIP-seq reads mapping to ms-snRNAs. (B) Relative distribution of ms-snRNA-mapping BCLIP-seq reads among individual snRNAs. (C) Northern blotting analysis of U1 and U2 snRNAs in immunoprecipitates of tandem affinity purified control untagged cells and indicated endogenously 3A-tagged cells. (D) BCLIP-seq signal coverage over U1 snRNA sequence (including reads mapping to U1a1, U1b1, and U1b6 isoforms) showing the two BCLIP-seq replicates of each sample separately. Gray columns indicate the positions of the Sm ring binding site and the four stem–loops within the U1 snRNA secondary structure. (E) CCDC174 BCLIP-seq signal coverage over U1 snRNA sequence (including reads mapping to U1a1, U1b1, and U1b6 isoforms) in WT and Nrde2-KO cells. (F) NRDE2 BCLIP-seq signal coverage over U1 snRNA sequence (including reads mapping to U1a1, U1b1, and U1b6 isoforms) in WT and SmE-dTAG cells. (G) Heat maps of the normalized NRDE2 BCLIP-seq signal intensity in WT and SmE-dTAG cells centered at annotated 5′SSs of all transcripts expressed in mESCs. Splice sites are ordered by decreasing NRDE2 signal in the WT sample, which is the same as in Figure 2C. (H) Schematic summary of the presented data showing that NRDE2 and CCDC174 associate with U1 snRNA to indiscriminately bind both strong and weak 5′SSs. Whereas depletion of either NRDE2 or CCDC174 has no effect on splicing strong 5′SSs, it leads to reduced splicing of weak 5′SSs with concomitant increase in intronic cryptic 5′SS usage.
