N6-methyladenosine modulates long non-coding RNA in the developing mouse heart

Long non-coding RNAs (lncRNAs) were reported to potentially play a regulatory role in the process of myocardial regeneration in the neonatal mouse. N6-methyladenosine (m(6)A) modification may play a key role in myocardial regeneration in mice and regulates a variety of biological processes through affecting the stability of lncRNAs. However, the map of m(6)A modification of lncRNAs in mouse cardiac development still remains unknown. We aimed to investigate the differences in the m(6)A status of lncRNAs during mouse cardiac development and reveal a potential role of m(6)A modification modulating lncRNAs in cardiac development and myocardial regeneration during cardiac development in mice. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) of the heart tissue in C57BL/6 J mice at postnatal day 1 (P1), P7 and P28 were performed to produce stagewise cardiac lncRNA m(6)A-methylomes in a parallel timeframe with the established loss of an intrinsic cardiac regeneration capacity and early postnatal development. There were significant differences in the distribution and abundance of m(6)A modifications in lncRNAs in the P7 vs P1 mice. In addition, the functional role of m(6)A in regulating lncRNA levels was established for selected transcripts with METTL3 silencing in neonatal cardiomyocytes in vitro. Based on our MeRIP-qPCR experiment data, both lncGm15328 and lncRNA Zfp597, that were not previously associated with cardiac regeneration, were found to be the most differently methylated at P1-P7. These two lncRNAs sponged several miRNAs which further regulated multiple mRNAs, including some of which have previously been linked with cardiac regeneration ability. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that differential m(6)A modifications were more enriched in functions and cellular signalling pathways related to cardiomyocyte proliferation. Our data suggested that the m(6)A modification on lncRNAs may play an important role in the regeneration of myocardium and cardiac development.