Fig. 2: ALKBH5 decreases the m6A abundance on MANF mRNA 3’-UTR.

A Pathway analysis of high/low MANF group of single cell sequencing data. B m6A sites predicted by SRAMP m6A prediction algorithm, showing 5 sites with high prediction confidence. C m6A pull-down assay assessing the m6A abundance of MANF in ACHN and 786-O cell lines with NC or ALKBH5 KO. Enrichment levels were normalized to NC group. D qRT-PCR detection of MANF expression level in in ACHN and 786-O cell lines with NC or ALKBH5 KO. Enrichment levels were normalized to NC group. E RIP assay assessing the binding of ALKBH5 on MANF mRNA and pre-mRNA in 786-O cell line. Enrichment levels were normalized to IgG, mRNA group. F RIP assay assessing the binding of ALKBH5 on MANF mRNA and pre-mRNA in ACHN cell line. Enrichment levels were normalized to IgG, mRNA group. G Actinomycin D assay assessing the stability of MANF mRNA in 786-O cell lines with NC or ALKBH5 KO. Levels were normalized to NC group, 0 h. H Actinomycin D assay assessing the stability of MANF mRNA in ACHN cell lines with NC or ALKBH5 KO. Levels were normalized to NC group, 0 h. I Upper: schematic diagram of MANF mRNA structure and the locations of three groups of m6A sites. Lower: m6A pull-down assay assessing three groups of m6A abundance of MANF in 786-O cell lines with NC or ALKBH5 KO. Enrichment levels were normalized to NC group. J Luciferase reporter assay of MANF point mutation in 293 cell lines with NC or ALKBH5 KO. Left: schematic diagram of MANF point mutation. Right: quantification of the Luciferase reporter assay. Levels were normalized to NC/MANF-WT group.