PTBP1 binds to CyCoNP to control the loading of miR-4492 on the lncRNA. (A) RT-qPCR quantification of PTBP1, CyCoNP, and NCAM1 transcripts in SK-N-BE cells (D 1.5) treated with si-SCR or si-PTBP1. Data were normalized to GAPDH mRNA and represent means ± SEM of four biological replicates. (B, left) Representative western blot analysis for PTBP1 and NCAM1 in SK-N-BE cells (D 1.5) treated with si-SCR or si-PTBP1. GAPDH was used as a loading control. (Right) Quantification of PTBP1 and NCAM1 signal intensities relative to GAPDH. Data represent means ± SEM of three biological replicates. (C, upper panel) Schematic representation of the CyCoNP luciferase-based reporter construct. The entire sequence of CyCoNP lncRNA was cloned downstream from the Renilla luciferase ORF (orange). The construct was cotransfected in SK-N-BE cells with a control siRNA (si-SCR) or siRNAs targeting PTBP1 (si-PTBP1). (Lower panel) Quantification of Renilla luciferase activity in SK-N-BE cells cotransfected with si-SCR or si-PTBP1. Data represent the mean luciferase activities ± SEM of four biological replicates. The relative position of miR-4492 MREs on the CyCoNP sequence is shown. (D, upper panel) Schematic representation of the CyCoNP ΔmiR-4492 luciferase-based reporter construct. (Lower panel) Quantification of Renilla luciferase activity in SK-N-BE cells cotransfected with the CyCoNP ΔmiR-4492 luciferase construct and a control siRNA (si-SCR) or siRNAs targeting PTBP1 (si-PTBP1). Data represent the mean luciferase activities ± SEM of four biological replicates. (E, upper panel) Schematic representation of CyCoNP endogenous RNA pull-down (PD). (Lower panel) RT-qPCR quantification of miR-4492 in CyCoNP PD RNA samples from SK-N-BE cells (D 1.5) treated with si-SCR or si-PTBP1. miR-1249-5p transcript serves as a negative control. Values are expressed as relative enrichment normalized over the % of CyCoNP transcript precipitated in each condition and represent means ± SEM of three biological replicates. (F, upper panel) Schematic representation of the CyCoNP ΔPTBP1 bs luciferase-based reporter construct. (Lower panel) Quantification of Renilla luciferase activity in SK-N-BE cells cotransfected with the CyCoNP ΔPTBP1 bs luciferase construct and a control siRNA (si-SCR) or siRNAs targeting PTBP1 (si-PTBP1). Data represent the mean luciferase activities ± SEM of four biological replicates. miR-4492 MREs relative position on CyCoNP sequence is shown. Data information: ns (nonsignificant) P > 0.05, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, unpaired Student's t-test.
