NRDE2 regulates 5′SS selection and splicing of its target gene Cdk2. (A) rMATS analysis (Shen et al. 2014) of alternative splicing events within NRDE2 target introns up-regulated (inclusion level difference >0.1) in Mtrex-KO cells and in CHX-treated WT and Nrde2-KO cells relative to their corresponding WT and untreated controls (only introns with more than 30 reads across all samples were included in the analysis). (B) UCSC genome browser screenshot showing normalized BCLIP-seq, RNA-seq, and Ribo-seq tracks over Cdk2 gene locus combined with a Sashimi plot of Cdk2 splicing patterns detected in the RNA-seq data. The thickness of the splice junction connecting lines reflects the number of corresponding spliced RNA-seq reads with a scale provided at the right. NRDE2 and CCDC174 target intron 1 is highlighted in red. (C) RT-PCR analysis of Cdk2 intron 1 splicing with primers binding in exons 1 and 3. The additional bands in Nrde2-KO sample were sequenced and the identified cryptic 5′SSs are depicted in the scheme at the top. (D) Schematic overview of the CDK2 dual-luciferase reporters. The point mutation introduced in Cdk2 intron 1 5′SS to improve U1 snRNA complementarity in the CDK2-strong5′SS reporter is depicted in red. (E,F) Dual-luciferase assay of the transiently transfected CDK2 reporters. Plotted is the average value of normalized Renilla luciferase activity from three independent experiments each performed in triplicate transfections. Error bars represent standard deviation. (G) RT-PCR analysis of CDK2 reporter splicing in transiently transfected WT and Nrde2-KO cells with primers binding in Cdk2 exon1 and in the P2A peptide region. The additional mutations introduced in the CDK2-wt5′SS reporter are depicted in red. The band numbering at the right corresponds to the scheme in C. (H) Same as in G, comparison of splicing in CDK2-wt5′SS reporter and CDK2-swap5′SS with the annotated 5′SS and the first cryptic 5′SS swapped, as depicted in the scheme.
