N6-Methyladenosine Methylome in Duchenne Muscular Dystrophy

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PROJECT SUMMARY/ABSTRACT Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease. Children with DMD become restricted to wheelchairs within the first decade of their lives and die within the third. There is no effective treatment available for this devastating disease. Key myogenic processes in DMD pathophysiology at the molecular level are not completely understood, hindering the development of therapies for DMD. To fill this knowledge gap, we have been investigating whether an emerging mode of post-transcriptional gene regulation, known as epitranscriptomics, plays a role in DMD pathogenesis. Epitranscriptomics is a dynamic process that regulates RNA stability, splicing, and translation by reversible chemical modifications of mRNAs. N6- methyladenosine (m6A) is the most abundant epitranscriptomic mark. The m6A marks are installed by methyltransferase like 3 (METTL3) and methyltransferase like 14 (METTL14) and erased by fat mass and obesity-associated (FTO) and alkylation repair homolog 5 (ALKBH5). Specific reader proteins selectively recognize m6A-modified mRNAs and control their fates. Our recent findings revealed that m6A mRNA methylation regulates myogenesis in DMD and that levels of the m6A writers, erasers, and m6A marks are tightly regulated during myoblast differentiation, muscle regeneration, and in DMD. These studies suggest that m6A mRNA methylation plays a pivotal role in DMD pathophysiology. Here, we propose to gain a deeper mechanistic insight into m6A mRNA methylation in DMD. We will perform a transcriptome-wide analysis to map m6A-modified mRNAs (methylome) in skeletal muscle stem cells (MuSCs), primary myoblasts, and skeletal muscle of a well- established mdx mouse model of DMD and its counterpart healthy mice. We will also map the m6A methylome in DMD patient-derived and healthy myoblasts to extend our conclusions to a more clinically relevant model system. We will mechanistically characterize a select subset of prioritized m6A target mRNAs, both previously associated with DMD, as well as the novel, to establish their link to muscle degeneration in DMD. Our proposed research will make a significant impact by revealing transcriptome-wide m6A target mRNAs in DMD and provide insights into DMD pathophysiology incurred by the dysregulation of this novel mechanism.