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Messenger RNA（mRNA）和核糖体RNA（RRNA）之间基本配对的范围和重要性的实验，作为调节真核生物翻译的机制。直到最近，这种机制仍被认为仅限于细菌。但是，通过使用相同的严格标准来建立细菌中的光泽 - 达尔加诺碱基配对相互作用，申请人表明，这种机制也会影响哺乳动物细胞的翻译。该演示使用了GTX同源域mRNA的5'领导者的9-NT转化增强子元件。 GFX元素的rRNA结合位点位于40S核糖体亚基的平台中，接近预测的mRNA结合道。申请人已经确定了许多其他转化增强子元素，并在18S rRNA中具有潜在的结合位点。这些站点主要位于40年代亚基的鞋间面上的两个离散区域：平台和右脚。拟议研究的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。紫外线交联与RNase H定位和锻炼方法一起评估和定义结合位点。基本配对的功能评估将涉及靶向具有互补寡核苷酸的18S rRNA中的候选位点，以及通过mRNA和rRNA序列的突变，以确定单个mRNA元素的活性是否需要与RRNA的完整互补匹配。在这些研究中，申请人开发了一种新型的酵母系统，其中含有小鼠杂种18S rRNA的核糖体。拟议研究的第二个主要目标是评估mRNA的生理相关性：rRNA相互作用。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，以确定在这种情况下是否发生碱基对以及这些相互作用是否影响蛋白质的合成。另外，将通过阻止和突变40S亚基中的结合位点评估这种碱基相互作用影响蛋白质组的程度，并测量这些突变对酵母和哺乳动物细胞中全球和特定蛋白质合成的影响。这些研究应提供有关如何调节mRNA的翻译的宝贵见解，从而允许控制翻译的新颖手段，例如用于生产治疗蛋白，以及分析疾病错误调节的潜在贡献的新途径。