Fig. 5.

METTL3/YTHDF3 complex increases the stability of MALAT1 in an m⁶A manner. a The m⁶A levels of MALAT1 from human lung cancer tissues were higher than their normal adjacent lung tissues (n = 10). b The RNA level of MALAT1 was analyzed by RT-PCR. c, d Sequence motifs in m⁶A peaks identified by using m6Avar database (http://m6avar.renlab.org/). e Putative m⁶A modification sites in the sequence of MALAT1 and synonymous mutations in the MALAT1. f–i qPCR analysis of immunoprecipitated m⁶A in A549 cells with transfection of indicated genes using MALAT1 PCR primer. j The relative m⁶A levels of MATAT1 Mut1-2 were detected in A549 using the MS2 system. k A549 cells were co-transfected with MALAT1-WT/Mut1-2 and METTL3 (left panel) or shMETTL3 (right panel), respectively. The expressions of MALAT1 were analyzed by qPCR. l, m qPCR shows that METTL3 dose-dependently increased the RNA levels of MALAT1-WT1 (l) but not of MALAT1-Mut1 (m) in A549 cells. n The relationship between the METTL3 and MALAT1 was analyzed basing on the TCGA database. o A549 and H1299 cells were co-transfected with Flag-YTHDF3/siYTHDF3 and MALAT1, respectively. The protein level of YTHDF3 was analyzed by WB from cellular lysate with immunoprecipitation of m⁶A antibody, respectively. p A549 and H1299 cells were co-transfected with Flag-YTHDF3 and indicated MALAT1 genes. The interactions between YTHDF3 and MALAT1 RNA were analyzed by RNA immunoprecipitation assay. q A549 cells were transfected with indicated genes. The m⁶A modifications of MALAT1 determined by using MeRIP-qPCR assay. r A549 cells were co-transfected with METTL3 WT/KD and siYTHDF3, respectively. The relative RNA level MALAT1 was analyzed by qPCR assay. Results were presented as mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups. NS, not significant