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Regulation of translation initiation in eukaryotes

真核生物翻译起始的调控

基本信息

批准号：
7993614
负责人：
VINCENT P MAURO
金额：
$ 16.32万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
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：
2009-12-25 至 2010-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7993614
关键词：
Affect Apoptosis Area Bacteria Base Pairing Base Sequence Be++ element Beryllium Binding Binding Sites Biochemical Bioinformatics Catalytic RNA Cell Cycle Cells Disease Elements Eukaryota Evaluation Event Face Genetic Enhancer Element Goals Hybrids Individual Initiator Codon Internal Ribosome Entry Site Length Mammalian Cell Measures Mediating Messenger RNA Methods MicroRNAs Mus Mutate Mutation Nucleotides Oligoribonucleotides Physiological Point Mutation Production Protein Biosynthesis Proteins Proteome RNA RNA Binding RNA Sequences RNA, Ribosomal, 16S RNA, Ribosomal, 18S Recombinants Regulation Reporter Research Personnel Ribonuclease H Ribosomal RNA Ribosomes Site Small Nuclear RNA System Testing Translating Translation Initiation Translations Yeasts biological adaptation to stress crosslink evidence base foot homeodomain insight novel programs research study therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): This proposal describes experiments to investigate the scope and importance of base-pairing between messenger RNA (mRNA) and ribosomal RNA (rRNA) as a mechanism for regulating translation in eukaryotes. Until recently, this mechanism was thought to be restricted to bacteria. However, by using the same rigorous criteria that were used to establish the Shine-Dalgarno base-pairing interaction in bacteria, the applicant has shown that this mechanism also affects translation in mammalian cells. This demonstration used a 9-nt translational enhancer element from the 5' leader of the Gtx homeodomain mRNA. The rRNA binding site for the Gfx-element is in the platform of the 40S ribosomal subunit, close to the predicted mRNA binding tract. The applicant has identified numerous other translational enhancer elements with potential binding sites in the 18S rRNA. These sites are primarily in two discrete regions in the intersubunit face of the 40S subunit: the platform and the right foot. A major goal of the proposed studies is to use both biochemical and functional approaches to determine if these sequences bind to rRNA and to determine if this binding affects translation. UV cross-linking together with RNase H localization and toeprinting methods will be used to evaluate and define binding sites. Functional evaluation of base-pairing will involve targeting candidate sites in the 18S rRNA with complementary oligoribonucleotides and by mutation of both mRNA and rRNA sequences to determine whether the activities of individual mRNA-elements require intact complementary matches to the rRNA. For these studies, the applicant has developed a novel yeast system with ribosomes containing a mouse-yeast hybrid 18S rRNA. The second major goal of the proposed studies is to assess the physiological relevance of mRNA:rRNA interactions. Natural mRNAs that contain ribosome binding sites will be tested in yeast and in mammalian cells to determine whether base-pairing occurs in this context and whether these interactions affect protein synthesis. In addition, the extent to which such base-pairing interactions affect the proteome will be assessed by blocking and mutating binding sites in 40S subunits and measuring the effects of these mutations on global and specific protein synthesis both in yeast and in mammalian cells. These studies should provide valuable insights into how the translation of mRNAs are regulated, allowing novel means for controlling translation, e.g. for the production of therapeutic proteins, and new avenues for analyzing the potential contributions of mis-regulation to disease.

描述（由申请人提供）：该提案描述了研究信使 RNA (mRNA) 和核糖体 RNA (rRNA) 之间碱基配对作为调节真核生物翻译机制的范围和重要性的实验。直到最近，这种机制还被认为仅限于细菌。然而，通过使用与在细菌中建立Shine-Dalgarno碱基配对相互作用相同的严格标准，申请人已经证明该机制也影响哺乳动物细胞中的翻译。该演示使用了来自 Gtx 同源域 mRNA 5' 前导序列的 9-nt 翻译增强子元件。 Gfx 元件的 rRNA 结合位点位于 40S 核糖体亚基的平台中，靠近预测的 mRNA 结合区。申请人已经鉴定出许多其他翻译增强子元件在18S rRNA中具有潜在的结合位点。这些位点主要位于 40S 亚基亚基间面的两个离散区域：平台和右脚。拟议研究的一个主要目标是使用生化和功能方法来确定这些序列是否与 rRNA 结合，并确定这种结合是否影响翻译。 UV 交联与 RNase H 定位和脚趾印迹方法一起用于评估和定义结合位点。碱基配对的功能评估将涉及用互补寡核糖核苷酸靶向 18S rRNA 中的候选位点，并通过 mRNA 和 rRNA 序列的突变来确定单个 mRNA 元件的活性是否需要与 rRNA 完整互补匹配。对于这些研究，申请人开发了一种新型酵母系统，其核糖体含有小鼠-酵母杂交 18S rRNA。拟议研究的第二个主要目标是评估 mRNA:rRNA 相互作用的生理相关性。含有核糖体结合位点的天然 mRNA 将在酵母和哺乳动物细胞中进行测试，以确定在此背景下是否发生碱基配对以及这些相互作用是否影响蛋白质合成。此外，通过阻断和突变 40S 亚基中的结合位点，并测量这些突变对酵母和哺乳动物细胞中整体和特定蛋白质合成的影响，可以评估这种碱基配对相互作用对蛋白质组的影响程度。这些研究应该为 mRNA 的翻译如何调控提供有价值的见解，从而提供控制翻译的新方法，例如用于生产治疗性蛋白质，以及分析错误调节对疾病的潜在影响的新途径。