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