Fig 2: ALKBH5-cIDR is required for hypoxia-induced m6A demethylation of NEAT1 and paraspeckles assembly.A, MeRIP (m6A-IP)-qPCR analysis of the NEAT1 m6A peak region in ALKBH5-deficient U87 cells rescued by A5-WT or dcIDR mutant under hypoxic conditions. Two-tailed unpaired Student’s t test. ∗p < 0.05, n = 3. B, real-time quantitative PCR analysis of RNA expression levels of NEAT1-2 in corresponding U87 cells under hypoxic conditions. Two-tailed unpaired Student’s t test, ∗∗, p < 0.01, n = 3. C, SFPQ immunostaining was performed in corresponding U87 cells under hypoxic conditions. Representative images are shown. The scale bars represent 2 μm. D, the number of paraspeckles was counted and analyzed in 20 corresponding U87 cells. Two-tailed unpaired Student’s t test, ∗∗∗p < 0.001.

Fig 3: Rapid ALKBH5 condensation is coupled with m6A demethylation of NEAT1 and paraspeckles assembly in response to hypoxia.A, ALKBH5 immunostaining was performed in NEAT1-deficient or control U87 cells after 1 h of hypoxia treatment. Representative images are shown. The scale bars represent 2 μm. B, MeRIP (m6A-IP)-qPCR analysis of the NEAT1 m6A peak region in U87 cells at different time points of hypoxia treatment. Two-tailed unpaired Student’s t test. ∗p < 0.05, n = 3. C, real-time quantitative PCR analysis of the RNA expression levels of NEAT1-2 in U87 cells at different time points of hypoxia treatment. Two-tailed unpaired Student’s t test. ∗, p < 0.05, ∗∗, p < 0.01, n = 3. D, SFPQ immunostaining was performed in U87 cells after 1 h of hypoxia treatment. Representative images are shown. The scale bars represent 2 μm.

Fig 4: Regulated expression levels of m6A-methylated mRNAs in mouse hippocampi after HL. (a) Time points for qRT-PCR and western blot analyses after inducing HL. (b) qRT-PCR analysis showed that the hippocampal mRNA expression levels of METTL14, WTAP, and YTHDF2 were significantly lower in the HL group than in the Con group and those of FTO and ALKBH5 were significantly higher. c, d) Western blotting results showed that the hippocampal protein expression levels of FTO and METTL3 were higher in the HL group than in the Con group, whereas those of METTL14 and YTHDF2 were comparable between both groups. The data shown were analyzed using an unpaired t-test and are presented as the mean ± SEM. Not significant (ns), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. n = 6 mice per group. Con, control; HL, hearing loss

Fig 5: Overview of m6A RNA methylation in mouse hippocampi after HL. (a) The number of peaks in each sample in both groups. An average of 26,780 and 25,958 peaks were detected in the Con and HL group, respectively. (b) Overlapping mRNA m6A peaks between the Con and HL groups are displayed with a Venn diagram. (c) The distribution of m6A peaks in the transcripts is displayed with a density curve for the 5′-UTR, CDS, and 3′-UTR. (d) The ratios of m6A-peak distributions in the Con and HL samples are displayed in pie charts. (e) Detection of a common motif enriched across the m6A peaks in both groups