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

Date Published: June 14, 2018

Publisher: Public Library of Science

Author(s): Jiayao Yu, Yi Li, Tian Wang, Xiang Zhong, Gabriele Multhoff.


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.

Partial Text

Heat shock proteins (HSPs), which are expressed constitutively in all species, are molecular chaperones that regulate protein localization, accumulation, and degradation, assist in protein re-folding, prevent protein aggregation [1], and play important physiological roles in normal conditions as well as situations involving both systemic and cellular stress [2, 3]. Based on their molecular sizes, HSP proteins are classified into a number of conserved protein families, including HSP90, HSP70, HSP60 and small HSPs [4]. It is well known that increase of HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1) [5]. Interestingly, growing evidence indicates that the expression of HSPs can be attributed to epigenetic changes including DNA methylation and acetylation of histones [6–9]. However, modulation of HSPs expression by RNA methylation remains unclear.

All the procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University, China.

As one of the most prevalent modifications in mRNAs, m6A methylation has emerged as a key post-transcriptional regulator of gene expression [23]. In the present study, we observed that METTL3 or YTHDF2 knockdown significantly changed HSPs gene expression, and heat shock induced nuclear localization of YTHDF2. M6A methylation peaks of HSPs are mainly enriched on exons and around stop codons. The precise mechanism for different expression of HSPs induced by m6A RNA methylation requires further investigation.




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