Fig. 9
SlALKBH2 protein binds SlDML2 transcript and promotes its stability by m6A demethylation. a RNA immunoprecipitation (RIP) assay showing that SlALKBH2 protein binds SlDML2 transcript. For the RIP assay, the protein-RNA complexes were extracted from wild-type tomato fruit at 42 days post-anthesis and subjected to immunoprecipitation with anti-ALKBH2 polyclonal antibody or rabbit IgG (negative control). Data are presented as mean ± standard deviation (n = 6). Asterisks indicate significant differences (***P < 0.001; Student’s t test). b Schematic of the transient expression system used for SlDML2 mRNA stability assay. The intact or mutated SlDML2 cDNA fragment, which is composed of coding sequence (CDS) and 3′ untranslated region (UTR), was cloned into pCambia2300 vector driven by the CaMV 35S promoter. The potential m6A modification site identified in m6A-seq was mutated from adenosine (A) to cytidine (C) using site-directed mutagenesis kit and highlighted in blue. c Determination of the SlDML2 mRNA stability. The intact (SlDML2-WT) or mutated SlDML2 (SlDML2-Mu) cDNA fragment was co-expressed with SlALKBH2-HA or empty vector control (HA) in Nicotiana benthamiana leaves. After actinomycin D treatment for 3 or 6 h, the total RNAs were isolated from the N. benthamiana leaves and submitted to quantitative RT-PCR assay. The N. benthamiana ACTIN gene was used as an internal control. Error bars represent the standard deviation of three independent experiments. d m6A-IP-qPCR assay showing the m6A enrichment in SlDML2 transcript. Data are presented as mean ± standard deviation (n = 3). Immunoblot analysis with anti-HA and anti-ALKBH2 antibodies shows the SlALKBH2 protein expression. Asterisks indicate significant differences (*P < 0.05; Student’s t test)