Functional Characterization of Two Novel Myofiber Type-Specific Non-histone Protein Methyltransferases

Chao Wang, Purdue University

Abstract

Skeletal muscles are composed of muscle cells or myofibers which are classified as four types (I, IIa, IIx/d, and IIb), with different physiological, biochemical, structural and metabolic characteristics. The molecular regulation of myofiber identify is not completely understood. I identified methyltransferase like 21 (Mettl21) e and Mettl21c as the type IIb and type I myofiber- specific protein, respectively. The aim of this dissertation was to determine the function of Mettl21e and Mettl21c in regulating myofiber identity. In the first part of the thesis, I used in silico approaches to identify Mettl21e as one of the top up-regulated genes in myostatin knockout (KO) mice, which exhibit robust muscle hypertrophy and increased proportion of IIb myofibers. Interestingly, the expression of Mettl21e is not directly regulated by myostatin, and its high level of expression in myostatin KO mice is a consequence of increased number of IIb myofibers. I then used gain-of-function and loss-of-function approaches to study the role of Mettl21e in muscle. Overexpression and knockdown of Mettl21e in cultured myoblasts increases and decreases the size of myotubes, respectively. Transgenic overexpression of Mettl21e in skeletal muscles of mice not only increases myofiber size of normally innervated muscles, but also ameliorates wasting of denervated muscles. By contrast, knockout of Mettl21e reduces the size of IIb myofibers without affecting fiber composition. Mass spectrometry reveals that Mettl21e interacts with subunits of 26S proteasome protein degradation system. Overexpression and knockout of Mettl21e suppresses and increases the activity of 26S proteasome, respectively. Notably, proteasome inhibitor rescues the size of Mettl21e-knockdown myotubes. These results establish a role of Mettl21e in increasing IIb myofiber size through decreasing proteasome activity. In the second part of the thesis, I focused on Mettl21c, a paralog of Mettl21e. I identified Mettl21c as a type I myofiber specific protein that tri-methylates heat shock protein 8 (Hspa8) at lysine-561 to increase its stability. The stabilized Hspa8 promotes selective degradation of MEF2A and MEF2D through chaperone-mediated autophagy. Overexpression of Mettl21c in myoblasts increases the level of Hspa8 and reduces the levels of MEF2A and MEF2D. Conversely, knockout of Mettl21c reduces the trimethylation of Hspa8 in type I-myofiber, leading to lower levels of Hspa8, higher levels of MEF2 proteins and increased expressions of slow muscle-specific genes. These results establish a role of Mettl21c in regulating type I myofiber homeostasis and function. In summary, my studies uncover the functions of two novel methyltransferases, extending the understanding of non-histone methyltransferases in biological process, and demonstrating the importance of myofiber type-specific proteins in regulating myofiber identity.

Degree

Ph.D.

Advisors

Kuang, Purdue University.

Subject Area

Molecular biology|Biochemistry|Developmental biology

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