Mechanism and Regulation of non-canonical translation initiation

核糖体是合成蛋白质的分子机器。 在蛋白质合成过程中，核糖体将信使核糖核酸（mRNA）序列解码为相应的蛋白质序列。 细胞mRNA起源于细胞染色体中的DNA。 核糖体不会随机地与mRNA结合;相反，有一个高度调节的过程来确保核糖体与特定位置的代码结合，以便mRNA代码正确地翻译成蛋白质。 虽然大多数细胞mRNA存在一个标准的起始途径，但在某些条件下（如应激），细胞中也存在替代途径。 了解这些替代途径也很重要，因为许多病毒都会利用它们。 该项目研究了一种称为内部起始的替代蛋白质生物合成途径。 内部启动需要RACK 1蛋白与核糖体结合，本项目将研究需要这种蛋白的原因。 通过替代途径翻译的mRNA也具有内部起始所需的结构特征。 拟议的工作将筛选代表数千种细胞mRNA的序列，以确定能够结合含有RACK 1的核糖体并允许内部翻译起始的RNA结构。这些方法将极大地提高我们的内部启动介导的蛋白质生物合成的组件和机制的理解。该研究计划由教育生活和学习计划补充，鼓励不同群体的本科生从事科学，技术，工程或数学职业。 生物学学生在这个程序的第一年生活，吃饭，一起学习，每周见面谈论他们的过渡到大学，寻找机会做研究与科学教师在他们的本科生涯早期，并在科学专业和职业生涯的成功策略。这项研究计划将有助于塑造下一代科学家。内部核糖体进入位点（IRES）是绕过mRNA的m7 G帽的要求以促进翻译的RNA结构。虽然病毒IRES已被深入研究，但细胞IRES序列还不太清楚。 为了研究细胞IRES介导的翻译的机制，将检查核糖体蛋白RACK 1与起始因子eIF 3D和DAP 5的相互作用，并鉴定通常由这些因子调节的RNA。在互补方法中，选择后在环状RNA构建体上起始的RNA序列将通过高通量测序鉴定，并在荧光素酶报告基因翻译测定中验证。这些数据将检验细胞IRES序列可能不限于5 ′非翻译区，而是可能在人类基因组中更广泛地发现的假设。总之，这些实验扩展了我们对蛋白质生物合成过程的知识，并为将来在单分子水平上进行蛋白质生物合成的机理研究奠定了基础。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

Ribosomes are molecular machines that synthesize proteins. During protein synthesis, ribosomes decode a messenger ribonucleic acid (mRNA) sequence into a corresponding protein sequence. Cellular mRNA originates from the DNA in a cell’s chromosomes. Ribosomes do not bind to mRNA at random; instead there is a highly-regulated process to ensure that ribosomes bind to the code in a particular place so that the mRNA code is translated correctly into protein. Although there is a standard pathway by which this initiation occurs for most cellular mRNAs, there are alternative pathways that occur in cells under certain conditions such as stress. It is also important to understand these alternative pathways because many viruses exploit them. This project investigates one alternative protein biosynthesis pathway known as internal initiation. Internal initiation requires the RACK1 protein to bind to the ribosome and this project will investigate the reasons this protein is required. The mRNA that is translated by alternative pathways also has structural features that are required for internal initiation. The proposed work will screen sequences representing thousands of cellular mRNAs to identify RNA structures that are able to bind ribosomes containing RACK1 and allow internal translation initiation. Together these approaches will greatly enhance our understanding of the components and mechanism of protein biosynthesis mediated by internal initiation. This research program is complemented by an educational living and learning program that encourages diverse groups of undergraduate students to pursue a career in science, technology, engineering, or mathematics. Biology students first years in this program live, eat, and study together and meet weekly to talk about their transition to college, finding opportunities to do research with science faculty early in their undergraduate careers, and strategies for succeeding in science majors and careers. This research program will help shape the next generation of scientists. Internal ribosomal entry sites (IRES) are RNA structures that bypass the requirement for the m7G cap of the mRNA to facilitate translation. Although viral IRESs have been studied intensively, cellular IRES sequences are less well-understood. To investigate the mechanism of cellular IRES-mediated translation, the interaction of the ribosomal protein RACK1 with the initiation factors eIF3D and DAP5 will be examined, and RNAs commonly regulated by these factors will be identified. In a complementary approach, RNA sequences initiating on a circular RNA construct following selection will be identified by high-throughput sequencing and validated in a luciferase reporter translation assay. These data will test the hypothesis that cellular IRES sequences may not be restricted to the 5ʹ untranslated region, but may be more widely found within the human genome. Together, these experiments expand our knowledge on the process of protein biosynthesis and lay the foundation to perform future mechanistic studies on protein biosynthesis at the single molecule level.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.