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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: Induction of m6A methylation in adipocyte exosomal LncRNAs mediates myeloma drug resistance

Fig. 6

METTL7A mediates RNA m6A methylation in LncRNAs. a. Transcriptome-wide mapping of MeRIP-seq shows the methylation profile of LOC606724 or SNHG1 in nADs or nADs exposed to MM ARP-1 or MM.1S cells. b. Mass spectrometry identifies the proteins in MMADs that were pulled down by biotin-labeled sense transcript of LOC606724 (LOC). RIP assay using anti-METTL7A antibodies shows the relative enrichment of LOC in the immunoprecipitates from MMAD lysates. Cell lysates pulled down by IgG served as control. c-f. Full length or truncated forms of His-tagged METTL7A were expressed and purified. c. Methyltransferase activity assay and dot blotting show the relative m6A levels in the mixture of various amount of His-tagged METTL7A and RNA substrates. d. Western blotting shows the full length (WT) and two truncated forms (â–³1 and â–³2) METTL7A protein immunoblotted with anti-His antibody and methyltransferase activity assays shows their relative m6A levels. e. Schematic illustration of seven putative METTL7A binding motifs and their respective custom RNA oligonucleotides (red or light red bar) on LOC transcript. Methyltransferase activity assay and dot blotting show relative m6A levels in the mixture of custom RNA oligonucleotides and full length METTL7A. f. Dot blotting shows relative m6A levels in the mixture of different combination of RNA oligonucleotides containing wild-type M5 motif (LOC M5) or mutated M5 motif (GGGCU, LOC M5AΔG) with full length or truncated METTL7A. g-l. MMADs were transfected with siRNA against METTL7A (siMETTL7A). Cells transfected with scrambled siRNA served as control (siCtrl). g. The levels of METTL7A proteins and mRNAs in siCtrl or siMETTL7A MMADs. h. Methyltransferase activity assay and dot blotting show relative m6A levels in siCtrl or siMETTL7A MMADs. i & j. RIP assay shows the relative enrichment of LOC or SNHG1 in the immunoprecipitates from lysates of siCtrl or siMETTL7A MMADs pulled down by anti-m6A antibodies (i) or by anti-hnRNPU or anti-hnRNPA2B1 antibodies (j). k. Quantitative real-time PCR analysis shows the relative level of exosomal LOC and SNHG1 derived from siCtrl or siMETTL7A MMADs. l. Annexin V-binding assay shows the percentages of apoptotic MM cells treated with 5 nM of bortezomib (BTZ) and exosomes derived from siCtrl or siMETTL7A MMADs. Treatment with vehicle served as control. In western blotting, GAPDH served as loading control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P values were determined by Student t-tests for comparison of two groups and one-way ANOVA for comparison of more than two groups

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