Expression of Muscle-Specific Ribosomal Protein L3-Like Impairs Myotube Growth

Abstract

The ribosome has historically been considered to have no cell-specific function but rather serve in a "housekeeping" capacity. This view is being challenged by evidence showing that heterogeneity in the protein composition of the ribosome can lead to the functional specialization of the ribosome. Expression profiling of different tissues revealed that ribosomal protein large 3-like (Rpl3l) is exclusively expressed in striated muscle. In response to a hypertrophic stimulus, Rpl3l expression in skeletal muscle was significantly decreased by 82% whereas expression of the ubiquitous paralog Rpl3 was significantly increased by ∼fivefold. Based on these findings, we developed the hypothesis that Rpl3l functions as a negative regulator of muscle growth. To test this hypothesis, we used the Tet-On system to express Rpl3l in myoblasts during myotube formation. In support of our hypothesis, RPL3L expression significantly impaired myotube growth as assessed by myotube diameter (-23%) and protein content (-14%). Further analysis showed that the basis of this impairment was caused by a significant decrease in myoblast fusion as the fusion index was significantly lower (-17%) with RPL3L expression. These findings are the first evidence to support the novel concept of ribosome specialization in skeletal muscle and its role in the regulation of skeletal muscle growth. J. Cell. Physiol. 231: 1894-1902, 2016. © 2015 Wiley Periodicals, Inc.

Figure 1

Rpl3l expression in adult mouse tissue and during skeletal muscle hypertrophy. A. Rpl3l and Rpl3 expression was determined by qPCR in several tissues of adult mice (n=4), normalized to Rpl38 expression. B. Microarray analysis of ribosomal protein gene expression during plantaris muscle hypertrophy induced by synergist ablation presented as fold-change relative to sham control; the microarray data accession number is GSE47098. C. The change in Rpl3l and Rpl3 expression observed by microarray during muscle hypertrophy was confirmed by qPCR; expression was normalized using the geometric mean based on the reference genes Rpl38, Gapdh and Ddit4. Data are expressed as mean +/− SE with significant (p < 0.05) difference from sham control (Sham) designated by asterisk.

Figure. 2

An in vitro model system to express RPL3L in C2C12 myogenic cells. A. Schematic of the pINDUCER Tet-On system used to express of RPL3L in C2C12 cells. Upon doxycycline (Dox) binding, constitutively expressed rtTA becomes activated with subsequent binding to the tetracycline response element (TRE), thereby inducing expression of HA-tagged RPL3L. B. Western blot showing exogenous RPL3L expression in myotubes in response to Dox treatment. RPL3L-C2C12 cells were differentiated four days in differentiation media containing 1 μg·mL⁻¹ Dox with RPL3L expression detected using a HA antibody. Alpha-tubulin was used as a loading control for Western blot analysis. C. Immunostaining showing the presence of exogenous RPL3L in myotubes in response to Dox treatment. RPL3L-C2C12 cells were differentiated four days in differentiation media with or without Dox (1 μg·mL⁻¹) and incubated with an antibody against HA. Nuclei were labelled by DAPI staining. D–E. RNA concentration and electrophoresis showing 18S and 28S rRNA following RPL3L-HA immunoprecipitation from 4-day differentiated myotubes treated with or without Dox (1 μg·mL⁻¹). F. Immunoblotting for exogenous RPL3L and RPS6 proteins from protein lysates and immunoprecipitate samples. TRAP: translating ribosome affinity purification.

Figure 3

RPL3L expression impairs myotube growth after four days of differentiation. A. Representative photographs from RPL3L-myotubes and empty vector (EV)-myotubes. B. Myotube diameter measured in RPL3L and EV myotubes. C. Protein content determined from protein lysates of RPL3L and EV myotubes. D. Quantification of the puromycin-labeled peptides in RPL3L-myotubes and representative image of immunoblot analysis followed by Coomassie Blue staining (loading control). Histograms represent mean ± SE from three biological replicates obtained in three independent experiments with significant difference (p < 0.05) from untreated RPL3L-myotubes designated by asterisk.

Fig. 4

RPL3L expression impairs myotube fusion after four days of differentiation. The fusion index was determined by counting the number of nuclei inside myosin heavy chain (MHC)-positive myotubes, divided by the total number of nuclei. Histograms represents mean ± SE from three biological replicates obtained in three independent experiments with significant difference (p < 0.05) from untreated RPL3L-myotubes designated by asterisk.