Figure 6.

METTL14 inhibits ESRP2 and Lnc-LSG1 interaction through YTHDC1

(A and B) Anti-YTHDC1 RIP assay showed that Lnc-LSG1 could significantly bind to YTHDC1. RIP, RNA immunoprecipitation (A). METTL14 can regulate the interaction between Lnc-LSG1 and YTHDC1 (B). (C and D) Anti-ESRP2 RIP assay showed increased interaction between Lnc-LSG1 and ESRP2 in shMETTTL14-1 cells and decreased interaction in OE-METTL14 cells (C). Knocking down YTHDC1 increased the binding between Lnc-LSG1 and ESRP2 (D), and blocked the inhibitory effect of OE-METTL14 on the interaction between Lnc-LSG1 and ESRP2 (D). (E). Anti-ESRP2 RIP assay showed that METTL14-WT, but not METTL14-R298P, could inhibit the interaction between ESRP2 and Lnc-LSG1. (F) Four RRACH motifs in the region 0–300 nt of Lnc-LSG1. (G) Schematic drawing of probe A and probe m⁶A. (H) RNA pull-down assay using probe A or probe m⁶A, followed by western blotting assay for ESRP2, YTHDC1, and GAPDH protein. (I) Western blotting assay was performed to investigate the effect of YTHDC1 on ESRP2 protein expression. (J) Western blotting assay showed that siYTHDC1 could partly reverse the Lnc-LSG1 overexpression-induced inhibition on ESRP2 expression. (K and L) siYTHDC1 partly blocked the effect of METTL14 overexpression on ESRP2 expression (K) and ubiquitination levels (L). Data are presented as the mean ± SD of at least three independent experiments.