Abstract
Increased ribosomal DNA transcription has been proposed to limit muscle protein synthesis, making ribosome biogenesis central to skeletal muscle hypertrophy. We examined the relationship between ribosomal RNA (rRNA) production and IGF-1–mediated myotube hypertrophy in vitro. Primary skeletal myotubes were treated with IGF-1 (50 ng/ml) with or without 0.5 µM CX-5461 (CX), an inhibitor of RNA polymerase I. Myotube diameter, total protein, and RNA and DNA levels were measured along with markers of RNA polymerase I regulatory factors and regulators of protein synthesis. CX treatment reduced 45S pre-rRNA expression (−64 ± 5% vs. IGF-1; P < 0.001) and total RNA content (−16 ± 2% vs. IGF-1; P < 0.001) in IGF-1-treated myotubes. IGF-1-mediated increases in myotube diameter (1.27 ± 0.09-fold, P < 0.05 vs. control) and total protein (+20 ± 2%; P < 0.001 vs. control) were not prevented by CX treatment. Suppression of rRNA synthesis during IGF-1 treatment did not prevent early increases in AKT (+203 ± 39% vs. CX; P < 0.001) and p70 S6K1 (269 ± 41% vs. CX; P < 0.001) phosphorylation. Despite robust inhibition of the dynamic ribosomal biogenesis response to IGF-1, myotube diameter and protein accretion were sustained. Thus, while ribosome biogenesis represents a potential site for the regulation of skeletal muscle protein synthesis and muscle mass, it does not appear to be a prerequisite for IGF-1-induced myotube hypertrophy in vitro.—Crossland, H., Timmons, J. A., Atherton, P. J. A dynamic ribosomal biogenesis response is not required for IGF-1–mediated hypertrophy of human primary myotubes.
Highlights
Ribosomal biogenesis is a coordinated, multistep process that plays a central biochemical role in cellular translational capacity and protein synthesis [1,2,3]
Transcription of 45S pre–ribosomal DNA by polymerase subunit A/B/C/E (RNA) polymerase I (Pol I) is considered a rate-limiting step in ribosomal biogenesis, which is in turn regulated by a number of key ABBREVIATIONS: CX, CX-5461; FOXO3a, forkhead box O 3a; LC3B, light chain 3B; MPS, muscle protein synthesis; mTOR, mammalian target of rapamycin; MuRF1, muscle RING finger 1; Pol I, polymerase I; POLR1A/ B/C/E RNA, polymerase 1 subunit A/B/C/E; Rb, retinoblastoma; rDNA, ribosomal DNA; RP, ribosomal protein; rRNA, ribosomal RNA; SL1, selectivity factor 1; TAF1A, TATA-box binding protein associated factor, RNA polymerase I, A; TIF1A, transcription initiation factor 1A; UBF, upstream binding factor
These events result in the activation of transcription initiation factor 1A (TIF1A), which interacts with Pol I and enables initiation of Pol I– dependent synthesis of 45S ribosomal RNA
Summary
Ribosomal biogenesis is a coordinated, multistep process that plays a central biochemical role in cellular translational capacity and protein synthesis [1,2,3]. Transcription of 45S pre–ribosomal DNA (rDNA) by RNA polymerase I (Pol I) is considered a rate-limiting step in ribosomal biogenesis, which is in turn regulated by a number of key ABBREVIATIONS: CX, CX-5461; FOXO3a, forkhead box O 3a; LC3B, light chain 3B; MPS, muscle protein synthesis; mTOR, mammalian target of rapamycin; MuRF1, muscle RING finger 1; Pol I, polymerase I; POLR1A/ B/C/E RNA, polymerase 1 subunit A/B/C/E; Rb, retinoblastoma; rDNA, ribosomal DNA; RP, ribosomal protein; rRNA, ribosomal RNA; SL1, selectivity factor 1; TAF1A, TATA-box binding protein associated factor, RNA polymerase I, A; TIF1A, transcription initiation factor 1A; UBF, upstream binding factor. C-Myc has been reported to increase after resistance exercise in animal models and humans [11, 12], and c-Myc may act as a regulator of the Pol I machinery [13] during human skeletal muscle hypertrophy
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