Ribosome specialization in adult skeletal muscle

成人骨骼肌的核糖体特化

• 批准号: 8822451
• 负责人: JOHN Joseph MCCARTHY
• 金额: $ 16.4万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822451
• 关键词: Ablation; Acquired Immunodeficiency Syndrome; Adult; Affect; Aging; Applications Grants; Atrophic; Biological Assay; Cachexia; Cells; Chronic; Clinical; Data; Databases; Deterioration; Development; Disease; EMSA; Embryonic Development; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Translation; Goals; Growth; HIV; Health; Heart failure; Heterogeneity; Housekeeping; Immobilization; In Vitro; Internal Ribosome Entry Site; Limb structure; Malignant Neoplasms; Mammals; Mediating; Messenger RNA; MicroRNAs; Molecular; Molecular Profiling; Morbidity - disease rate; Mus; Muscle; Muscle Fibers; Muscle function; Mutagenesis; Myogenic Regulatory Factors; Nature; Pattern; Peptidyltransferase; Play; Process; Proteins; Quality of life; Reading; Regulation; Reporter; Repression; Rheumatoid Arthritis; Ribosomal Frameshifting; Ribosomal Proteins; Ribosomes; Risk; Role; Sepsis; Skeletal Muscle; Striated Muscles; System; Systemic disease; Terminator Codon; Testing; Tetanus Helper Peptide; Tissues; Transcript; Transfer RNA; Translating; Translations; Untranslated Regions; base; effective therapy; indexing; interest; loss of function; mortality; muscle form; muscle hypertrophy; neurotensin mimic 1; novel; patient population; prevent; programs; promoter; public health relevance; research study; response; ribosomal protein L3; sarcopenia; skeletal muscle plasticity

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.

描述（由申请人提供）：核糖体通常被认为不具有细胞特异性功能，而是起到“管家”的作用。这一观点受到了证据的挑战，证据显示核糖体蛋白质组成的异质性导致了核糖体的功能特化。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。成人组织中核糖体蛋白的表达谱表明，核糖体蛋白L3（Rpl3）在除横纹肌外的所有组织中均高表达；相反，Rpl3-like的表达恰恰相反，仅在横纹肌、尤其是骨骼肌中高表达。初步数据显示，在重大时期 肌肉肥大，例如出生后发育以及对增效剂消融的反应，这两个基因的表达模式完全逆转，使得骨骼肌中 Rpl3 高表达而 Rpl3 样显着下调。与这些观察结果一致，我们发现 RPL3 样的过度表达会促进与较低融合指数相关的显着肌管萎缩。虽然 Rpl3 对于核糖体组装和核糖体肽基转移酶活性是必需的，但 Rpl3 样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。为了检验骨骼肌中核糖体特化及其调节的想法，以下目标 我们将致力于：1) 确定肌肉特异性 Rpl3-like 如何改变核糖体功能，2) 确定调节 Rpl3 和 Rpl3-like 肌肉特异性表达模式的机制。我们开发了一种 Tet-On 系统来比较含有 Rpl3 的核糖体与 Rpl3 样核糖体的翻译保真度。通过评估程序化核糖体移码、终止密码子通读、近源和非同源 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，在使用 miR-1 模拟物和 antimiR 的功能获得和丧失研究中进行诱变，检验肌肉特异性 microRNA-1 抑制骨骼肌中 Rpl3 表达的假设， 分别。接下来，我们将通过启动子分析、诱变、EMSA 和 ChIP 检测来检验 MyoD 调节 Rpl3-like 肌肉特异性表达的假设。存在肌肉特异性 Rpl3 的事实强烈表明核糖体已经专门针对骨骼肌 - 了解为什么会这样是该项目的长期目标。短期目标是更好地了解其功能和调节Rpl3 样的 为未来 R01 拨款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基础。