Modification of N6-methyladenosine RNA methylation on heat shock protein expression

This study was conducted to investigate effect of N6-methyladenosine (m6A) RNA methylation on Heat shock proteins (HSPs) and dissect the profile of HSP RNA methylation. The results showed that m6A methyltransferases METTL3 mRNA was decreased in responses to heat shock stress in HepG2 cells, but m6A-specific binding protein YTHDF2 mRNA was upregulated in a manner similar to HSP70 induction. Immunofluorescence staining showed that the majority of YTHDF2 was present in the cytosol, however, nearly all YTHDF2 translocated from the cytosol into the nucleus after heat shock. METTL3 knockdown significantly changed HSP70, HSP60, and HSP27 mRNA expression in HepG2 cells using siRNA, however, mRNA lifetime was not impacted. Silence of YTHDF2 using siRNA did not change expression of HSP70, but significantly increased HSP90, HSP60, and HSPB1 mRNA expression. In addition, m6A-seq revealed that HSP m6A methylation peaks are mainly enriched on exons and around stop codons, and shows a unique distribution profile in the 5'UTR and 3'UTR. Knockdown of METTL3 changed the methylation patterns of HSPs transcript. In conclusion, m6A RNA methylation regulates HSP gene expression. Differential expression of HSPs modulated by m6A may depend on the m6A site and abundance of the target gene. This finding provides insights into new regulatory mechanisms of HSPs in normal and stress situations.