Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Decoding the cardiac epitranscriptome using Nanopore sequencing

N. Díaz i Pedrosa1, L. M. Weiß1, A. Schneider2, M. Staps2, T. Braun2, R. Gilsbach1

1Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität - Fachbereich Medizin, Frankfurt am Main; 2Max-Planck-Institut für Herz- und Lungenforschung, Bad Nauheim;

RNA modifications are key regulators of RNA stability, thus regulating gene expression at a posttranscriptional level. N6-Methyladenosine (m6A) is the most studied RNA modification in the cardiovascular field. It is known to decorate essential transcripts for the cardiac linage, but the exact position of the modifications remains unknown. The establishment of m6A is performed by the Methyltransferaselike 3 (Mettl3) RNA-specific enzyme. Direct RNA sequencing (DRS) using nanopores is a third-generation sequencing technique that allows us to identify cardiac RNA modifications (the epitranscriptome) with base pair resolution. In this work, we combine DRS and cardiomyocyte-specific Mettl3 KO mice to identify m6A sites. After performing DRS on ventricles from WT and Mettl3 KO mice, we obtain a sequencing depth of over 1,5 Mio reads in average per sequencing run (WT1 2,45 Mio, WT2 1,81 Mio, KO1 1,15 Mio and KO2 1,63 Mio). These data identify more than 1.800 significantly modified RNA regions in the cardiac transcriptome. Integration of data from cardiomyocyte-specific Mettl3 KOs reveal that more than 22% of the modified sites detected by DRS in cardiomyocyte-specific transcripts are sensitive to ablation of Mettl3 and thus represent m6A sites. Remarkably, we observe that transcripts losing m6A modifications are upregulated. In contrast, expression of non-cardiomyocyte transcripts, which are not affected by the cardiomyocyte-specific deletion, are not altered. In conclusion, direct RNA sequencing using nanopores allows us to detect RNA modifications in an unbiased manner with base pair resolution. Integration of Mettl3 KO data identifies m6A modifications in cardiomyocyte transcripts. Our data suggest that m6A plays a role in posttranscriptional gene expression regulation in cardiomyocytes via promoting mRNA degradation, as transcripts losing the modification tend to have higher expression values.

https://dgk.org/kongress_programme/jt2022/aP1939.html