Fig. 3

NEAT1–1 interacted with CYCLINL1 and CDK19 through m6A site #4. a and b Upper, protein diagrams with RNA recognition motif (rrm) were shown. Lower, CLIP-qPCR analyswas of Flag or Myc binding at the NEAT1 in P-18 out cells transfected with Flag-CDK19-WT, Flag-CDK19-mut (rrm deletion), Myc-CYCLINL1-WT or Myc-CYCLINL1-muts (rrms deletion). Nature mutations were shown in the diagrams. PKinase, protein kinase domain. All data shown were mean values ± SD (error bar) from three replicates. P values were shown in the figures. c and d CLIP-qPCR analyswas of CDK19 or CYCLINL1 binding at the NEAT1–1 in P-18 cells with different primers targeting different regions. e RNA EMSA evaluation of CDK19 or CYCLINL1 binding of NEAT1–1 RNA probes. Flag-CDK19 or Myc-CYCLINL1 recombinant proteins were produced using Quick coupled transcription/translation kit through T7 promoter in vitro. Flag-CDK19 or Myc-CYCLINL1 recombinant proteins were incubated with biotin-labeled in vitro transcribed NEAT1–1 probes in the presence or absence of m6A modification (single nucleotide “A” deletion (3494 nt of NEAT1–1)), followed by PAGE and immune blotting with HRP-conjugated streptavidin. f Co-IP of endogenous CYCLINL1 or CDK19 with CDK19 or CYCLINL1 from P-18 cell lysate pre-treated with RNaseA in 37 °C for 30 min. The protein complexes were detected by western blot