Ribosome specialization in adult skeletal muscle

描述(申请人提供)：核糖体通常被认为没有细胞特有的功能，而是起到“管家”的作用。这一观点受到了核糖体蛋白质组成异质性的证据的挑战，这导致了核糖体的功能特化。核糖体专门化改变了核糖体的内在翻译活性，从而影响特定的mRNAs ET的翻译。核糖体蛋白在成人组织中的表达谱显示，核糖体蛋白L3(Rpl3)在除横纹肌以外的所有组织中都高表达；相反，Rpl3样蛋白的表达正好相反，只在横纹肌，特别是骨骼肌中高表达。初步数据显示，在显著增长的时期 肌肉肥大，如出生后发育和对增效剂消融的响应，这两个基因的表达模式完全颠倒，使Rpl3在骨骼肌中高表达，Rpl3样蛋白显著下调。与这些观察一致，我们发现RPL3样蛋白的过度表达促进了与较低融合指数相关的显著的肌管萎缩。虽然Rpl3是核糖体组装和多肽转移酶活性所必需的，但Rpl3样蛋白的核糖体功能仍然完全未知。根据我们的初步数据，我们假设Rpl3样蛋白通过改变骨骼肌核糖体的内在翻译活性来限制肌肉大小。为了测试骨骼肌核糖体特化的想法及其调控，以下目标 将继续研究：1)确定肌肉特异性Rpl3样蛋白如何改变核糖体功能；2)确定调节肌肉特异性Rpl3和Rpl3样蛋白表达模式的机制。我们已经开发了一个Tet-on系统来比较含有Rpl3的核糖体和类似Rpl3的核糖体的翻译保真度。核糖体功能将通过评估程序性核糖体框架移位、停止密码子通读、近同源和非同源tRNA利用以及IRES介导的翻译来检查。在初步数据的基础上，我们将分别使用miR-1模拟和抗miR的获得和功能丧失研究中的32-UTR报告基因突变来验证肌肉特异性microRNA-1抑制Rpl3在骨骼肌中表达的假设。接下来，我们将通过启动子分析、突变、EMSA和芯片检测来验证MyoD调控肌肉特异性Rpl3样蛋白表达的假设。存在肌肉特异的Rpl3这一事实强烈表明核糖体已经成为骨骼肌的专门化-了解为什么会这样是该项目的长期目标在短期内，目标是更好地理解Rpl3-like作为 为将来的R01拨款申请奠定基础，调查核糖体特化在骨骼肌可塑性和疾病中的作用。

DESCRIPTION (provided by applicant): 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.