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. 2021 Jul 28;7(31):eabg7073. doi: 10.1126/sciadv.abg7073

Fig. 3. The m6A modification expedites the degradation of HMBOX1 mRNA per se, which is mediated by the reader protein YTHDF2.

Fig. 3

(A) Levels of specified mRNA molecules in LNCaP cells transfected with control siRNA (siCtrl) or siRNA targeting METTL3 (siM3) for 72 hours. (B and C) Half-life of HMBOX1 mRNA in LNCaP cells upon silencing METTL3 (B) or ALKBH5 (C) with the treatment of ActD (5 μg/ml) before total mRNAs were collected at indicated time points. (D) Levels of indicated molecules upon YTHDF2 knockdown in LNCaP. (E) Expression of HMBOX1 in the control (siCtrl) and YTHDF2-knockdown (siDF2) cells of LNCaP stably expressing empty backbone (vector), wild-type METTL3 (M3-WT), or catalytically dead mutant (M3-CD). (F) Schematic illustration of effective (A) and noneffective (A-10nt) sgRNA targeting HMBOX1 transcript (sgHMBOX1). (G) MeRIP-qPCR analysis of m6A signals on HMBOX1 in LNCaP cells expressing dCas9 fused to the wild-type ALKBH5 (dCas9-ALKBH5-WT) or to the incompetent demethylase (dCas9-ALKBH5-H204A) together with control sgRNA (sgCtrl), sgHMBOX1-A, or sgHMBOX1-A-10nt. GAPDH was included as a negative control. (H) Expression of HMBOX1 in the CRISPR-dCas9–based, m6A-editing system that is described in (G). (I) Half-life of HMBOX1 mRNA in LNCaP cells expressing dCas9-ALKBH5-WT together with specified sgRNAs and treated with ActD as described in (B) and (C).