Identification of differentially m6A methylated circRNAs in patient derived RBM20 mutated and control iPS-Cardiomyocytes

by Emma Katharina Hummel
Doctoral thesis
Date of Examination:2025-05-07
Date of issue:2025-04-30
Advisor:Prof. Dr. Karl Toischer
Referee:Prof. Dr. Karl Toischer
Referee:Prof. Dr. André Fischer
Persistent Address: http://dx.doi.org/10.53846/goediss-11243

 

 

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Abstract

English

Approximately 5% of dilated cardiomyopathies are caused by a mutation in the RBM20 gene, which encodes a protein involved in the splicing of cardiac specific titin and formation of cTTNs, the titin specific circRNA. Although circular RNAs (circRNAs) belong to the class of non-coding RNAs, recent findings suggest that they can indeed be translated independently of their 3′ and 5′ ends. The functions of proteins encoded by circRNAs remain largely unknown. Among various epigenetic modifications, N6-methyladenosine (m6A methylation) is the most prevalent in both DNA and RNA, and has been shown to enhance the translation efficiency of circRNAs. Each of these elements, RBM20 mutations, circRNA biogenesis, and m6A methylation, has been individually implicated in the development of heart failure. However, no studies to date have examined their interactions and potentially combined impact on cardiac dysfunction. In this study, we isolated and characterized m6A-methylated circRNAs from induced pluripotent stem cell (iPSC)-derived cardiomyocytes from RBM20 mutated patients and healthy controls, using MeRIP (methylated RNA immunoprecipitation) and subsequent next-generation sequencing. We also analyzed the expression of enzymes responsible for m6A methylation at both gene and protein levels using qPCR and Western blotting. Our results revealed a higher abundance of m6A-methylated circRNAs in RBM20 mutated cardiomyocytes compared to controls. Additionally, the m6A modification sites within circRNAs differed significantly between the two groups. The general transcriptional activity appeared elevated in RBM20 mutated cells. Furthermore, we identified PTPRZ1, PAK3, and NTRK2 as the most prevalent m6A-modified circRNAs unique to RBM20 mutated cells. Interestingly, the expression of m6A methylation-related enzymes on gene level was significantly higher in control cells, while the expression on protein level was significantly elevated in the RBM20 mutated cells. This study is the first to investigate the interplay between RBM20 mutations and m6A methylation in circRNAs, providing novel insights into their potential role in the pathogenesis of heart failure.
Keywords: circRNA; m6A methylation; heart failure; RBM20