Nontranslational function of leucyl-tRNA synthetase regulates myogenic differentiation and skeletal muscle regeneration
Kook Son, … , Sunghoon Kim, Jie Chen
Kook Son, … , Sunghoon Kim, Jie Chen
Published May 1, 2019; First published April 15, 2019
Citation Information: J Clin Invest. 2019;129(5):2088-2093. https://doi.org/10.1172/JCI122560.
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Concise Communication Muscle biology Therapeutics

Nontranslational function of leucyl-tRNA synthetase regulates myogenic differentiation and skeletal muscle regeneration

Abstract

Aside from its catalytic function in protein synthesis, leucyl-tRNA synthetase (LRS) has a nontranslational function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also regulates skeletal myogenesis, but the signaling mechanism is distinct from that in cell growth regulation. A role of LRS in myogenesis has not been reported. Here we report that LRS negatively regulated myoblast differentiation in vitro. This function of LRS was independent of its regulation of protein synthesis, and it required leucine-binding but not tRNA charging activity of LRS. Local knock down of LRS accelerated muscle regeneration in a mouse injury model, and so did the knock down of Rag or Raptor. Further in vitro studies established a Rag-mTORC1 pathway, which inhibits the IRS1-PI3K-Akt pathway, to be the mediator of the nontranslational function of LRS in myogenesis. BC-LI-0186, an inhibitor reported to disrupt LRS-Rag interaction, promoted robust muscle regeneration with enhanced functional recovery, and this effect was abolished by cotreatment with an Akt inhibitor. Taken together, our findings revealed what we believe is a novel function for LRS in controlling the homeostasis of myogenesis, and suggested a potential therapeutic strategy to target a noncanonical function of a housekeeping protein.

Authors

Kook Son, Jae-Sung You, Mee-Sup Yoon, Chong Dai, Jong Hyun Kim, Nidhi Khanna, Aditi Banerjee, Susan A. Martinis, Gyoonhee Han, Jung Min Han, Sunghoon Kim, Jie Chen

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Figure 1

LRS negatively regulates myogenesis in a translation-independent manner.

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LRS negatively regulates myogenesis in a translation-independent manner....
(A) C2C12 cells were transduced with lentiviruses expressing shLRS or shScramble (control), puromycin-selected for 2 days, and differentiated for 72 hours, followed by measurement of differentiation index as described in the Supplemental Methods. Data were normalized to shScramble (n = 3). (B) Cells treated as in A were lysed every 24 hours and subjected to Western blotting analysis (n = 3). (C) Cells were treated as in A, and at 0 and 24 hours of differentiation, EdU incorporation was conducted for 2 hours as described in the Supplemental Methods (n = 3). (D) TA muscles were coinjected with BaCl2 and lentiviruses expressing shLRS or shScramble, and isolated on day 7 after injury (D7AI) (n = 5) and day 14 after injury (D14AI) (n = 6), followed by measurement of cross-sectional area (CSA) of regenerating myofibers as described in the Supplemental Methods. Data were presented as the size distribution of all myofibers with average CSA. (E) Cells were transfected with Myc-LRS-WT, F50A/Y52A, K716A/K719A, or empty vector (EV; control), selected with G418, and differentiated for 72 hours, followed by measurement of differentiation index. Data were normalized to EV (n = 3). (F) Cells were either transduced with lentiviruses expressing shLRS or shScramble, followed by puromycin-selection for 2 days (black bars), or treated with different concentrations of cycloheximide (CHX) for 24 hours (gray bars). Confluent cells were subjected to measurement of protein synthesis rate by [35S]Met/Cys metabolic labeling (n = 4). *P < 0.05, **P < 0.01 by 1-way ANOVA (A) or 2-tailed paired t test (D). The data in E and F denoted by #, §, ‡ are significantly different from each other by 1-way ANOVA (P < 0.05). All error bars represent SEM.