Fig. 2

Upregulation of circRIMS2 is mediated through METTL3-dependent m6A modification. a The abundance of m6A-modified circRIMS2 was analyzed by MeRIP-PCR in the hippocampus of 4-month-old WT and APP/PS1 mice (n = 3). b The protein levels of ALKBH5, METTL3, YTHDC1, and IGF2BP1 were detected by WB, and quantitative analysis was performed (n = 3). c The positions of circRIMS2 sequence-based m6A modification sites were predicted using SRAMP (http://www.cuilab.cn/sramp) (upper). Sequence validation of the m6A modification sites of WT, Mut1, and Mut2 in circRIMS2 mRNA was performed by Sanger sequencing (lower left). qRT-PCR analysis of circRIMS2 was conducted after transfection with WT, Mut1, and Mut2 plasmids in N2a cells (lower right) (n = 5, one-way ANOVA with Tukey’s post-hoc test). d RNA antisense purification (RAP) was performed to screen the binding proteins of circRIMS2 (left). Specific peptide fragments of METTL3 were identified by mass spectrometry (right). e The abundance of m6A-modified circRIMS2 was analyzed by MeRIP-PCR in N2a cells after overexpressing METTL3 (n = 3). f The abundance of m6A-modified circRIMS2 between the vector- and METTL3-overexpressing N2a cells with WT, Mut1, or Mut2 plasmid transfection by MeRIP-PCR (n = 3, one-way ANOVA with Tukey’s post-hoc test). g qRT-PCR analysis of circRIMS2 was conducted in N2a cells treated with ActD for 4 h after transfection with shControl or shMETTL3 (n = 6, one-way ANOVA with Tukey’s post-hoc test). Data are presented as mean ± SEM and two-tailed t tests were used unless otherwise specified. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT in b, vs Vector group in c and e, vs Vector+WT group in f. ^#P < 0.05, ^###P < 0.001 vs WT in c; vs the METTL3 + WT group in f; vs the shControl + ActD group in g