NRDE2 and CCDC174 bind to pre-mRNA 5′SSs. (A) Schematic workflow of the BCLIP-seq protocol. Template-switching oligo (TSO). (B) Mapping characteristics of the BCLIP-seq reads. The category “rRNA” contains reads mapping to mouse rDNA (GenBank: BK000964), category “mRNA” comprises reads mapping to protein-coding mature mRNAs and category “other” includes all remaining reads mapping to the mouse genome. The two bars in each sample represent two independent BCLIP-seq replicates. (C) Heat maps of the normalized BCLIP-seq signal intensity centered at annotated 5′SSs of all transcripts expressed in mESCs. Splice sites are ordered by decreasing NRDE2 signal. (D) Metaplots of normalized NRDE2, CCDC174, and EIF4A3 BCLIP-seq signal around all annotated expressed 5′SSs. (E) Violin plot showing ratios of spliced versus unspliced NRDE2, CCDC174, and EIF4A3 BCLIP-seq reads spanning annotated 5′SSs. Only 5′SSs of introns longer than 1 kb with a minimum of 10 splice site-overlapping reads were included in the analysis. White dots and bars represent the mean and standard deviation of all analyzed introns. (F) Density plots of the distribution of NRDE2, CCDC174, and EIF4A3 BCLIP-seq signal on all expressed introns ranked by their 5′SS strength, 3′SS strength, branch point strength, intron length, and GC content. (G) Metaplots of normalized NRDE2, CCDC174, and EIF4A3 BCLIP-seq signal around 5′SSs of introns selected based on their 5′SS, 3′SS, and branch point strength. Solid and dashed lines represent signal on introns with all three features above and below median strength, respectively.
