N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA and long noncoding RNAs. m6A is induced by the Mettl3 complex most commonly near stop codons and 3? UTRs in mRNAs. m6A recruits the YTH domain family of m6A-binding proteins, which then control almost every aspect of RNA metabolism, including alternative splicing, nuclear export, stability, and translation. Several thousand mRNA regions are modified by m6A in a given cell population, primarily at the consensus sequence of DRACH (D=A, G, or U; R=A or G; H=A, C, or U). Reflecting the diverse molecular functions of m6A, it also regulates a wide range of biological phenomena, such as cancer cell proliferation, neural development, and pluripotent stem cell differentiation. However, the roles of m6A in muscle cell differentiation remain largely elusive. The PI?s group recently found that one of the Fkbp family member of peptidyl prolyl isomerases interacts with the Mettl3 complex. In addition, depletion of the Fkbp inhibited myoblast differentiation and decreased the total level of m6A in the cells, both of which were recapitulated by Mettl3 depletion. At a molecular level, the Fkbp promotes cis-trans isomerization of Mettl3 and its related protein Mettl14 in vitro. Based on these findings, they hypothesized that the Fkbp regulates the m6A level of myoblast mRNAs through isomerization of Mettl3 and Mettl14.
Three aims were proposed to investigate this hypothesis.
Aim 1 will determine genome-wide distribution of m6A in myoblasts at a single nucleotide level and study how the pattern changes by depletion of the Fkbp.
Aim 2 will characterize how m6A affects metabolism of several most abundantly modified myoblast-specific mRNAs.
Aim 3 will study how Fkbp8 regulates Mettl3 and Mettl14, focusing on phosphorylation and isomerization of the proteins. These studies are expected to lay a solid foundation for a future study of the roles of m6A in muscle cells.
This project will study how the peptidyl prolyl isomerase Fkbp regulates mRNA methylation that are important for muscle cell differentiation. It can contribute to improve our understanding of muscle development and regeneration from a novel mechanistic perspective.