The ribosome has generally been considered to have no cell-specific function but rather serves in a "housekeeping" capacity. This view has been challenged by evidence showing heterogeneity in the protein composition of the ribosome, resulting in the functional specialization of the ribosome. Ribosome specialization alters the intrinsic translational activity of the ribosome thereby affecting the translation of a particular et of mRNAs. Expression profiling of ribosomal proteins in adult tissues showed that ribosomal protein L3 (Rpl3) is highly expressed in all tissues except in striated muscle;in contrast, the expression of the Rpl3-like is exactly the opposite, being highly expressed only in striated muscle, skeletal muscle in particular. Preliminary data revealed that during periods of significant muscle hypertrophy, such as post-natal development and in response to synergist ablation, the expression pattern of these two genes is completely reversed such that Rpl3 is highly-expressed and Rpl3-like is dramatically down-regulated in skeletal muscle. Consistent with these observations, we found that over-expression of RPL3-like promotes significant myotube atrophy associated with a lower fusion index. While Rpl3 is necessary for ribosome assembly and peptidyl transferase activity of the ribosome, the ribosomal function of Rpl3-like remains completely unknown. Based on our preliminary data, we hypothesize that Rpl3-like acts to limit muscle size by altering the intrinsic translational activity of skeletal muscle ribosomes. To test the idea of ribosome specialization in skeletal muscle and its regulation, the following objectives will be pursued: 1) Determine how muscle-specific Rpl3-like alters ribosome function and 2) Determine the mechanisms regulating the muscle-specific pattern of expression of Rpl3 and Rpl3-like. We have developed a Tet-On system to compare translational fidelity of ribosomes containing Rpl3 versus Rpl3- like. Ribosome function will be examined by assessing programmed ribosomal frameshifting, stop codon read- through, near- and non-cognate tRNA utilization and IRES-mediated translation. Based on preliminary data, we will test the hypothesis that the muscle-specific microRNA-1 represses Rpl3 expression in skeletal muscle by using 32-UTR reporter assay with mutagenesis in gain- and loss-of-function studies using miR-1 mimic and antimiR, respectively. Next, we will test the hypothesis that MyoD regulates the muscle-specific expression of Rpl3-like through promoter analysis, mutagenesis, EMSA and ChIP assay. The fact that there is a muscle- specific Rpl3 strongly suggests ribosomes have become specialized in skeletal muscle - understanding why this is so is the long-term goal of this project In the short-term, the goal is to better understand the function and regulation of Rpl3-like as the basis for a future R01 grant application investigating the role of ribosome specialization in skeletal muscle plasticity and disease.
The loss of skeletal muscle mass is of clinical importance because it is associated with increased morbidity and mortality as well as a marked deterioration in the quality of life. A broad patient population is affected by significant losses n muscle mass including those afflicted by various systemic diseases (cancer, sepsis, HIV- AIDS, heart failure), chronic physical inactivity, rheumatoid arthritis, limb immobilization and sarcopenia. A better of understanding of the molecular mechanisms that regulate skeletal muscle mass will provide the basis for more effective therapies to prevent or restore muscle mass. The focus of the current proposal is to investigate the regulation and function of ribosome specialization in adult skeletal muscle and the role it plays in skeletal muscle health and disease.