Fig. 4

YTHDC1 promotes cytoplasmic export of m⁶A methylated circNSUN2. a Left, identification of the circNSUN2-protein complex pulled down by circNSUN2 junction probe with protein extracts from HCT116 cells. The arrows indicating the additional band presented in circNSUN2-protein complex. Right, immunoblot analysis of YTHDC1 after pulldown assay showing its specific association with circNSUN2. b RIP assays showing the association of YTHDC1 with circNSUN2. Top, IP efficiency of YTHDC1-antibody shown in western blotting. Bottom, relative enrichment representing RNA levels associated with YTHDC1 relative to an input control. IgG antibody served as a control. Data represent mean ± S.D. from three independent experiments; dot plot reflects data points from independent experiment. c MeRIP assay showing that circNSUN2 was highly recruited in m⁶A precipitated fraction. Data represent mean ± S.D. from three independent experiments; dot plot reflects data points from independent experiment. The P values were determined by a two-tailed paired Student’s t test. d Top, schematic illustration showing the GAACU m⁶A motif located at exon 5−exon 4 junction site of circNSUN2. Bottom, the sequence of RNA probe for RNA-EMSA assay. e RNA-EMSA assay showing the binding ability of purified YTHDC1 with biotin-labeled oligonucleotides containing GAACU motif from circNSUN2. f Cytoplasmic and Nuclear mRNA Fractionation experiment showing that knockdown of YTHDC1 increased the nuclear circNSUN2 content, whereas the dysregulation of nuclear circNSUN2 caused by YTHDC1 RNAi was recovered by overexpression of WT but not the mutant of YTHDC1. Data represent mean ± S.D. from five independent experiments; dot plot reflects data points from independent experiment. The P values were determined by a two-tailed unpaired Student’s t test. g RNA-FISH showing that the increased nuclear staining of circNSUN2 caused by YTHDC1 RNAi was rescued by overexpression of WT, but not the mutant YTHDC1. Scale bar, 10 µm. Data represent mean ± S.D. from three independent experiments; dot plot reflects data points from independent experiment. The P values were determined by a two-tailed unpaired Student’s t test. Source data are provided as a Source Data file. Unprocessed original scans of blots are shown in Supplementary Fig. 10