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Fig. 8 | Genome Biology

Fig. 8

From: RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

Fig. 8

SlALKBH2 is a demethylase for mRNA m6A demethylation in tomato. a Sequence alignment of the highly conserved AlkB domain in SlALKBH2, Arabidopsis ALKBH9B (AtALKBH9B), and mouse ALKBH5 (MmALKBH5B). Fe (II) binding sites and alpha-ketoglutaramate (α-KG) binding sites are highlighted by green and red rectangles, respectively. b A proposed reaction mechanism of oxidative demethylation of N6-methyladenosine (m6A) to adenosine (A) by SlALKBH2. c Recombinant SlALKBH2 protein directly demethylates the m6A modification in m6A-containing ssRNA in vitro. The digested substrates cytidine (C), uridine (U), guanosine (G), A, and m6A were analyzed by HPLC. d SlALKBH2 demethylates m6A modification in mRNA in vivo. Endogenous mRNA was isolated from Nicotiana benthamiana leaves transiently expressing the SlALKBH2-HA fusion protein or the empty plasmid control (HA) and used for LC-MS/MS assay. Data are presented as mean ± standard deviation (n = 3). Asterisks indicate significant differences (*P < 0.05; Student’s t test). Immunoblot analysis was performed to detect SlALKBH2 expression using both anti-HA and anti-ALKBH2 antibodies. e Subcellular localization showing that SlALKBH2 locates in the endoplasmic reticulum (ER). His-Asp-Glu-Leu (HDEL) represents an ER retention signal peptide. Protoplasts of the N. benthamiana leaves transiently expressing eGFP alone or co-expressing ALKBH2-eGFP and HDEL-RFP were isolated and observed under a Leica confocal microscope (Leica, DMI600CS). Scale bar = 50 μm

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