Regulation of translation initiation in eukaryotes

描述(申请人提供)：本提案描述了调查信使RNA(信使RNA)和核糖体RNA(RRNA)之间碱基配对作为调节真核生物翻译的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，申请人已经表明，这种机制也影响哺乳动物细胞的翻译。本演示使用了来自Gtx同源域基因5‘端的9-NT翻译增强子元件。GFX元件的rRNA结合位点位于40S核糖体亚基的平台上，接近预测的mRNA结合区。申请人已经确定了在18S rRNA中具有潜在结合位点的许多其他翻译增强子元件。这些部位主要位于40年代亚基的亚基间面上的两个离散区域：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。将使用UV交联法以及RNaseH定位和脚印方法来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苷酸靶向18S rRNA中的候选位点，并通过突变mRNA和rRNA序列来确定单个mRNA元件的活性是否需要与rRNA完整的互补匹配。在这些研究中，申请人开发了一种新的酵母系统，其核糖体含有小鼠-酵母杂交的18S rRNA。拟议研究的第二个主要目标是评估mRNA：rRNA相互作用的生理学相关性。含有核糖体结合位点的天然mRNAs将在酵母和哺乳动物细胞中进行测试，以确定碱基配对是否在这种情况下发生，以及这些相互作用是否影响蛋白质合成。此外，这种碱基配对相互作用对蛋白质组的影响程度将通过阻断和突变40S亚基的结合位点，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来评估。这些研究应该为如何调控mRNAs的翻译提供有价值的见解，为控制翻译提供新的手段，例如生产治疗性蛋白质，并为分析错误调控对疾病的潜在贡献提供新的途径。

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.