m6A methyltransferase RBM15 promotes cardiomyocytes survival under hypoxia by increasing Thbs4 RNA methylation and mediating PI3K/Akt signaling

Abstract Background mRNA modifications constitute ancient mechanisms in regulating gene expression after transcription. N6-methyladenosis (m6A), which is the most prevalent internal RNA modification, is not only installed by m6A methyltransferases, removed by demethylases, but also specifically bounded by RNA-binding proteins. As a significant component in the m6A methyltransferase complex, RNA binding motif protein 15 (RBM15) plays a vital role in m6A methylation. Nevertheless, its function and mechanism in myocardial infarction (MI) remain poorly defined. Purpose To investigate the role and mechanism of RBM15 in regulating its targets through m6A methylation in MI. The research results will not only add new content to the basic mechanism of myocardial protection but also provide new ideas and new targets for the prevention and treatment of MI. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to clarify the total m6A level, and Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot were used to determine the expression of RBM15 in normal and MI tissues. Then the effects of RBM15 on cardiomyocytes were clarified using apoptosis assay, and cell proliferation assay. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), and transcriptomic RNA sequencing (RNA-seq) were used to perform the regulator mechanism of RBM15 on target gene Thbs4 in MI. Results In this research, we showed that total m6A methylation was increased in MI, and RBM15 was a main factor involved with this process. Silencing RBM15 remarkably decreased cell proliferation and increased apoptosis in vitro, and resulted in severe cardiac remodeling and further exacerbation of cardiac dysfunction in vivo, whereas its overexpression caused the opposite effects. Then, Thbs4 was identified as a direct downstream target of RBM15, and RBM15 induced m6A methylation on the 3'UTR of Thbs4 pre-mRNA. We also found that it showed faster Thbs4 mRNA decay and exhibited decreased mRNAs and levels of protein expression in RBM15-deficient cardiomyocytes under hypoxia. Furthermore, we confirmed that RBM15 contributed significantly to regulate the PI3k/Akt pathway. Conclusions Our work uncovers a complex RBM15-Thbs4-PI3K/Akt regulatory model based on m6A methylation and provides a new insight into the epi-transcriptomic dysregulation in MI development. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China