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Quality Control in Ribosome Assembly - the Function of Regulatory Proteins

核糖体组装的质量控制 - 调节蛋白的功能

基本信息

批准号：
9767230
负责人：
Katrin Karbstein
金额：
$ 53.21万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-10 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767230
关键词：
Address Archaea Aspartate Binding Binding Sites Biochemical Biogenesis Biological Assay Bypass Cells Colorectal Cancer Coupled Data Data Set Development Diamond-Blackfan anemia Disease Dissociation Ensure Event Failure Funding Genetic Genomics Goals Grant Human Incidence Individual Lead Link Luciferases Malignant Neoplasms Malignant neoplasm of liver Malignant neoplasm of prostate Messenger RNA Molecular Monitor Outcome Pathway interactions Patients Phenotype Phosphorylation Phosphotransferases Physiologic pulse Point Mutation Production Proteins Quality Control Reagent Reporter Ribosomal Proteins Ribosomal RNA Ribosomes Role Series Sodium Chloride Specificity Stress Structure Syndrome Terminator Codon Testing Translating Translations Work Yeasts cancer cell chromosome 5q loss endonuclease experimental study factor A genetic regulatory protein human disease in vivo insight malignant breast neoplasm mutant novel programs protein protein interaction proteostasis transcriptome sequencing translational model yeast genetics

项目摘要

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.

项目摘要快速分裂的细胞必须每分钟产生2,000个新的核糖体，并确保它们功能齐全不完全组装的核糖体逃逸到翻译池中这是Diamond Blackfan患者癌症发病率高的原因贫血、5q综合征与先天性无脾。我们工作的一个重点是质量控制机制在40S亚基组装的后期阶段起作用。在在这项资助下，我们将专注于最后一个核糖体蛋白Rps26是如何被整合到核糖体，它在翻译过程中的作用是什么，以及它的缺失对蛋白质的影响体内平衡此外，该分析将扩展到另外两种核糖体蛋白，描述mRNA通道中的全套接触。在目标1中，我们将剖析最后一个在40S成熟的步骤中，组装因子Nob1和Pno1的释放，通过 Rio1激酶。因为Rps26和Pno1具有高度重叠的结合位点，所以Rps26和Pno1的释放是可能的。 Pno1是Rps26掺入所必需的。我们将利用遗传和生化实验，以Rio1和ATP依赖的方式探测Nob1和Pno1的释放，并测试其作用从旁路的Rio1激酶对核糖体保真度。在目标2中，我们将通过以下方式定义机制：哪些Rps26缺陷的核糖体产生，Rps26在翻译中的作用是什么，终止和拖延是。这些目标将通过体内脉冲追踪来实现实验，来自不同酵母菌株的核糖体中Rps26水平的分析，荧光素酶报告基因测定和核糖体足迹法。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测定，以分析两种基因的mRNA特异性。不同的r-蛋白缺陷核糖体，即缺少Rps0和Rps10的核糖体。通过分析这三个数据集（包括Rps26）在一起，我们将不仅定义在r-protein-mRNA的接触，整个mRNA结合通道，而且还破译了不同的r-蛋白的单倍不足导致了两种共有的以及高度不同的表型。这些实验需要在上一个供资期间生产的优势调查结果和试剂，并使用独特的遗传学，基因组学和生物化学实验相结合，以解决这些基本的问题.