Ribosome specialization in adult skeletal muscle

描述（由申请人提供）：核糖体通常被认为没有细胞特异性功能，而是具有“管家”能力。这一观点受到了证据的挑战，这些证据表明核糖体的蛋白质组成具有异质性，导致核糖体的功能特化。核糖体特化改变了核糖体的内在翻译活性，从而影响特定mRNA的翻译。成年组织中核糖体蛋白的表达谱显示，核糖体蛋白L3（Rpl 3）在除横纹肌之外的所有组织中高度表达;相反，Rpl 3样蛋白的表达正好相反，仅在横纹肌，特别是骨骼肌中高度表达。初步数据显示，在重大时期， 肌肉肥大，如出生后发育和对骨骼肌切除的反应，这两种基因的表达模式完全逆转，使得Rp 13在骨骼肌中高度表达，Rp 13样在骨骼肌中显著下调。与这些观察结果一致，我们发现RPL 3样的过表达促进与较低融合指数相关的显著肌管萎缩。虽然Rpl 3是核糖体组装和核糖体的肽基转移酶活性所必需的，但Rpl 3样的核糖体功能仍然完全未知。基于我们的初步数据，我们假设Rpl 3样通过改变骨骼肌核糖体的内在翻译活性来限制肌肉大小。为了验证骨骼肌核糖体特化及其调节的想法，以下目标 将继续：1）确定肌肉特异性Rpl 3样如何改变核糖体功能和2）确定调节Rpl 3和Rpl 3样的肌肉特异性表达模式的机制。我们已经开发了Tet-On系统来比较含有Rpl 3与Rpl 3样的核糖体的翻译保真度。将通过评估程序性核糖体移码、终止密码子通读、近同源和非同源tRNA利用以及IRES介导的翻译来检查核糖体功能。基于初步数据，我们将通过使用32-UTR报告基因测定，分别使用miR-1模拟物和antimiR在功能获得和功能丧失研究中进行诱变，来检验肌肉特异性microRNA-1抑制骨骼肌中Rp 13表达的假设。接下来，我们将通过启动子分析、诱变、EMSA和ChIP测定来检验MyoD调节Rpl 3样的肌肉特异性表达的假设。存在肌肉特异性Rpl 3的事实强烈表明核糖体已经在骨骼肌中变得特化-理解为什么会这样是该项目的长期目标。 为将来R 01基金申请研究核糖体特化在骨骼肌可塑性和疾病中的作用奠定了基础。

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.