Leucine Differentially Regulates Gene-Specific Translation in Mouse Skeletal Muscle

Background: Amino acids, especially leucine, are particularly effective in promoting protein synthesis. Leucine is known to increase the rate of protein synthesis in skeletal muscle through the mechanistic target of rapamycin complex 1-dependent, as well as -independent, signaling pathways. However, the overall translation program is poorly defined, and it is unknown how the activation of these pathways differentially controls the translation of specific mRNAs.Objective: Ribosome profiling and RNA sequencing were used to precisely define the translational program activated by an acute oral dose of leucine.Methods: Adult male C57BL/6 mice were deprived of food overnight before the delivery of an acute dose of l-leucine (9.4 mg) (n = 6) or vehicle (n = 5) and tissues collected 30 min later. Ribosome footprints and total RNA were isolated and subjected to deep sequencing. Changes in gene-specific mRNA abundance and ribosome occupancy were determined between the leucine-treated and control groups by aligning sequence reads to Reference Sequence database mRNAs and applying statistical features of the Bioconductor package edgeR.Results: Our data revealed mRNA features that confer translational control of skeletal muscle mRNAs in response to an acute dose of leucine. The subset of skeletal muscle mRNAs that are activated consists largely of terminal oligopyrimidine mRNAs (false discovery rate: <0.05), whereas those with reduced translation had 5' untranslated regions with increased length. Only the small nuclear RNAs, which are required for ribosome biogenesis, were significantly altered in RNA abundance. The inferred functional translational program activated by dietary leucine includes increased protein synthesis capacity and energy metabolism, upregulation of sarcomere-binding proteins, modulation of circadian rhythm, and suppression of select immune components.Conclusions: These results clarify the translation program acutely stimulated by leucine in mouse skeletal muscle and establish new methodologies for use in future studies of skeletal muscle disease or aging and further examination of downstream effects of leucine on gene expression.