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中文摘要
翻译
项目总结 快速分裂的细胞必须每分钟产生2000个新的核糖体,并确保它们 功能齐全。未完全组装的核糖体逃逸到翻译池 钻石黑粉患者癌症发病率高的原因 贫血、5q综合征和先天性脾功能不全。我们的工作重点之一就是剖析 在40s亚单位组装后期阶段运行的质量控制机制。在……里面 在这项资助中,我们将重点研究最后一种核糖体蛋白Rps26是如何被结合到 核糖体,它在翻译中的作用是什么,以及它的缺失对蛋白质的影响 动态平衡。此外,这一分析将扩展到另外两种核糖体蛋白,以 描述在信使核糖核酸通道中的全套接触。在目标1中,我们将剖析其中一个决赛 在40s成熟的步骤中,通过活性释放组装因子Nob1和Pno1 核糖核酸激酶。由于Rps26和Pno1具有高度重叠的结合位点,因此释放 Pno1是Rps26的整合所必需的。我们将利用基因和生化实验来 以Rio1和ATP依赖的方式检测Nob1和Pno1的释放,并检测其效果 核糖体保真度上的Rio1激酶旁路。在目标2中,我们将通过以下方式定义这些机制 产生了哪些Rps26缺失的核糖体,以及Rps26在翻译中的作用 终止和拖延是。这些目标将通过体内的脉搏追逐来实现 不同酵母菌核糖体中Rps26水平的实验分析 记者分析和核糖体足迹。最后,在目标3中,我们将使用核糖体纯化, Rna-seq分析和荧光素酶报告基因分析 明显的r蛋白缺陷型核糖体,缺乏rps0和rps10。通过分析这三个方面 数据集(包括Rps26)一起,我们将不仅定义r-蛋白质-mRNA接触 整个mRNA结合通道,但也破译了不同r-蛋白单倍体不足的原因 导致了共同的和高度不同的表型。这些实验需要 在上一次资助期间产生的优势结果和试剂,并使用独特的 结合遗传学、基因组学和生物化学实验来解决这些基本问题 问题。
英文摘要
PROJECT SUMMARY Rapidly dividing cells must produce 2,000 new ribosomes every minute, and ensure that they are fully functional. Escape of incompletely assembled ribosomes into the translating pool underlies the high cancer incidence observed in patients suffering from Diamond Blackfan Anemia, 5q- syndrome and congenital asplenia. One focus of our work has been to dissect quality control mechanisms operational during late stages of assembly of the 40S subunit. In this grant, we will focus on how the last ribosomal protein, Rps26, is incorporated into ribosomes, what its roles during translation are, and the consequences of its absence on protein homeostasis. Furthermore, this analysis will be expanded to two additional ribosomal proteins to describe the full set of contacts in the mRNA channel. In Aim 1, we will dissect one of the final steps in 40S maturation, the release of the assembly factors Nob1 and Pno1, via the activity of the Rio1 kinase. Because Rps26 and Pno1 have a highly overlapping binding site, release of Pno1 is required for incorporation of Rps26. We will use genetic and biochemical experiments to probe the release of Nob1 and Pno1 in a Rio1 and ATP-dependent manner, and test the effect from bypass of the Rio1 kinase on ribosome fidelity. In Aim 2, we will define the mechanisms by which Rps26-deficient ribosomes are produced, and what the role of Rps26 in translation termination and stalling is. These goals will be accomplished through in vivo pulse-chase experiments, analysis of Rps26 levels in ribosomes from different yeast strains, luciferase reporter assays and ribosome footprinting. Finally, in Aim 3, we will use ribosome purifications, RNA-seq analysis, and luciferase reporter assays to analyze the mRNA-specificity of two distinct r-protein-deficient ribosomes, those lacking Rps0 and Rps10. By analyzing these three datasets (including Rps26) together, we will define not just the r-protein-mRNA contacts in the entire mRNA binding channel, but also decipher how haploinsufficiency of different r-proteins leads to both shared as well as highly divergent phenotypes. These experiments take advantage findings and reagents produced during the last funding period, and use a unique combination of genetics, genomics and biochemical experiments to address these fundamental questions.
期刊论文(20)
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会议论文
DOI: 10.1016/j.cbpa.2011.07.023
发表时间: 2011-10
期刊: CURRENT OPINION IN CHEMICAL BIOLOGY
影响因子: 7.8
作者: [Karbstein, Katrin]
通讯作者: Karbstein, Katrin
DOI: 10.1016/j.tcb.2013.01.004
发表时间: 2013-05
期刊: TRENDS IN CELL BIOLOGY
影响因子: 19
作者: [Karbstein, Katrin]
通讯作者: Karbstein, Katrin
Protein-protein interactions within late pre-40S ribosomes.
40S 前后期核糖体内的蛋白质-蛋白质相互作用。
DOI: 10.1371/journal.pone.0016194
发表时间: 2011-01-20
期刊: PloS one
影响因子: 3.7
作者: [Campbell MG, Karbstein K]
通讯作者: Karbstein K
DOI: 10.1083/jcb.201804163
发表时间: 2018-12-03
期刊: The Journal of cell biology
影响因子: --
作者: [Collins JC, Ghalei H, Doherty JR, Huang H, Culver RN, Karbstein K]
通讯作者: Karbstein K
12
    Dissecting the Mechanisms of Regulation and Quality Control in Ribosome Assembly and the Consequences of their Failure
    • 批准号:
      10162623
    • 项目类别:
    • 资助金额:
      $33.52万
    • 财政年份:
      2020
    • 负责人:
      Katrin Karbstein
    • 依托单位:
    Dissecting the Mechanisms of Regulation and Quality Control in Ribosome Assembly and the Consequences of their Failure
    • 批准号:
      10640194
    • 项目类别:
    • 资助金额:
      $95.21万
    • 财政年份:
      2020
    • 负责人:
      Katrin Karbstein
    • 依托单位:
    Dissecting the Mechanisms of Regulation and Quality Control in Ribosome Assembly and the Consequences of their Failure
    • 批准号:
      10406314
    • 项目类别:
    • 资助金额:
      $95.21万
    • 财政年份:
      2020
    • 负责人:
      Katrin Karbstein
    • 依托单位:
    Dissecting the Mechanisms of Regulation and Quality Control in Ribosome Assembly and the Consequences of their Failure
    • 批准号:
      10601284
    • 项目类别:
    • 资助金额:
      $60.2万
    • 财政年份:
      2020
    • 负责人:
      Katrin Karbstein
    • 依托单位:
    海外基金