Transient Focal Ischemia Significantly Alters the m6A Epitranscriptomic Tagging of RNAs in the Brain

Abstract

Background and Purpose- Adenosine in many types of RNAs can be converted to m⁶A (N⁶-methyladenosine) which is a highly dynamic epitranscriptomic modification that regulates RNA metabolism and function. Of all organs, the brain shows the highest abundance of m⁶A methylation of RNAs. As recent studies showed that m⁶A modification promotes cell survival after adverse conditions, we currently evaluated the effect of stroke on cerebral m⁶A methylation in mRNAs and lncRNAs. Methods- Adult C57BL/6J mice were subjected to transient middle cerebral artery occlusion. In the peri-infarct cortex, m⁶A levels were measured by dot blot analysis, and transcriptome-wide m⁶A changes were profiled using immunoprecipitated methylated RNAs with microarrays (44 122 mRNAs and 12 496 lncRNAs). Gene ontology analysis was conducted to understand the functional implications of m⁶A changes after stroke. Expression of m⁶A writers, readers, and erasers was also estimated in the ischemic brain. Results- Global m⁶A levels increased significantly at 12 hours and 24 hours of reperfusion compared with sham. While 139 transcripts (122 mRNAs and 17 lncRNAs) were hypermethylated, 8 transcripts (5 mRNAs and 3 lncRNAs) were hypomethylated (>5-fold compared with sham) in the ischemic brain at 12 hours reperfusion. Inflammation, apoptosis, and transcriptional regulation are the major biological processes modulated by the poststroke differentially m⁶A methylated mRNAs. The m⁶A writers were unaltered, but the m⁶A eraser (fat mass and obesity-associated protein) decreased significantly after stroke compared with sham. Conclusions- This is the first study to show that stroke alters the cerebral m⁶A epitranscriptome, which might have functional implications in poststroke pathophysiology. Visual Overview- An online visual overview is available for this article.

Keywords: RNA Methylation; apoptosis; inflammation; m6A demethylase.

Fig. 1:. Cerebral ischemia elevated global m⁶A levels.

Dot blot quantification of m⁶A abundance in the mRNA isolated from the cortical peri-infarct region of male mice at 3h, 6h, 12h and 24h of reperfusion (A) and female mice at 24h of reperfusion (B) following 1h transient MCAO. Data are mean±SEM (n=5/group). 1 to 5 above each blot represents individual animals. *p<0.05 compared with sex-matched sham by Mann-Whitney U test.

Fig. 2:. Cerebral ischemia significantly altered m⁶A epitranscriptome.

Principal component analysis of differentially m⁶A modified transcripts showed distinct clusters of stroke (12h reperfusion following 1h transient MCAO) and sham groups (n =5/group) (A). The percentage of variance associated with PC1 and PC2 are 80.1 and 5.4 respectively. Amplification of positive (Pos) and negative (Neg) spike-in RNA controls by real-time PCR showed comparable MeRIP enrichment between stroke (12h reperfusion following 1h transient MCAO) and sham groups (B). Fold change was calculated by normalizing %(MeRIP/Input) of Pos with Neg control for corresponding groups. Data are mean±SEM (n=5/group). *p<0.05 compared with sham by Mann-Whitney U test. Volcano plots show mRNAs (C) and lncRNAs (D) that are m⁶A hypermethylated (red dots) and hypomethylated (blue dots) in stroke group (12h reperfusion following 1h transient MCAO) over the sham group (n=5/group). The green threshold lines are set at 5-fold (p<0.05) between ischemia and sham.

Fig. 3:. Real-time PCR confirmed ischemia-induced differential m⁶A enrichment in RNAs.

MeRIP-qPCR was used to confirm microarray data for 4 hypermethylated (A) and 4 hypomethylated transcripts (B) in stroke samples (12h reperfusion following 1h transient MCAO) compared to sham samples. Fold change was calculated by normalizing %(MeRIP/Input) of stroke samples with sham samples. Tnf-α, tumor necrosis factor α; Sele, E-selectin; Hspa1a, heat shock protein a1a; Gpr34, G protein-coupled receptor 34; Caly, Neuron-specific vesicular protein calcyon; Slc39a10, solute carrier family 39 member 10. RP23-99G21.3 and RP23.340N6.1 are lncRNA transcripts. The mRNA levels of m⁶A readers YTHDF1 (C) and YTHDF3 (D) increased significantly in the peri-infarct cortex of male mice at 24h of reperfusion following 1h transient MCAO compared to sham. Data are mean±SEM (n=3–5/group). PCR was conducted in triplicate. *p<0.05 compared with sham by Mann-Whitney U test.

Fig. 4:. Differentially m⁶A modified transcripts participate in stroke pathophysiology.

GO analysis for the molecular function and biological process, and KEGG pathway analysis of post-stroke differentially m⁶A modified mRNA transcripts.The X-axis is the gene counts and the number next to each bar is the percent enrichment of a particular GO category.

Fig. 5:. Cerebral ischemia curtailed FTO expression and activity.

Following 1h transient MCAO, FTO mRNA levels evaluated by real-time PCR were significantly down-regulated at 3h, 6h, 12h and 24h of reperfusion compared to sham (A). FTO protein levels evaluated by western blotting (B) and demethylase activity analyzed by ELISA (C) were also significantly decreased at 12h and 24h of reperfusion following transient MCAO compared to sham. Peri-infarct cortical tissue from adult male mice was used in these experiments. Data are mean±SEM (n=3–5/group). *p<0.05 compared with sham by Mann-Whitney U test.

Fig. 6:. FTO protein is predominantly expressed in neurons.

In sham-operated mice, FTO immunostaining was identified mostly in the NeuN⁺ neurons (A) than in GFAP⁺ astrocytes (B) and IBA1⁺ microglia (C). In the peri-infarct cortex of stroke (12h of reperfusion after 1h transient MCAO) samples, FTO protein levels decreased significantly in the neu rons. DAPI, 4’,6-diamidino-2-phenylindole. Scale bar is 30 μm.