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)序列解码成相应的蛋白质序列。细胞信使核糖核酸来源于细胞染色体中的DNA。核糖体并不是随机地与信使核糖核酸结合；相反，有一个高度调控的过程来确保核糖体与特定位置的密码结合，从而使信使核糖体密码正确翻译成蛋白质。虽然对于大多数细胞mRNAs来说，这种启动有一个标准的途径，但在某些条件下，如应激，细胞中也有其他途径发生。了解这些替代途径也很重要，因为许多病毒都会利用它们。这个项目研究了一种可供选择的蛋白质生物合成途径，称为内部启动。内部启动需要RACK1蛋白与核糖体结合，该项目将调查需要该蛋白的原因。通过其他途径翻译的信使核糖核酸也具有内部启动所需的结构特征。这项拟议的工作将筛选代表数千个细胞mRNAs的序列，以识别能够与含有RACK1的核糖体结合并允许内部翻译启动的RNA结构。总之，这些方法将极大地提高我们对内部引发介导的蛋白质生物合成的组成和机制的理解。这一研究计划与教育生活和学习计划相辅相成，鼓励不同群体的本科生在科学、技术、工程或数学领域追求职业生涯。生物专业的学生在这个项目的第一年一起生活、吃饭和学习，每周都会聚在一起，谈论他们到大学的过渡，在他们本科生涯的早期找到机会与理科教师一起做研究，以及在理科专业和职业生涯中取得成功的策略。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mRNAm7G帽的要求的RNA结构，以促进翻译。虽然病毒IRESS已被深入研究，但对细胞IRES序列了解较少。为了研究细胞IRES介导的翻译机制，我们将研究核糖体蛋白RACK1与启动因子eIF3D和DAP5的相互作用，并确定这些因子共同调控的RNA。在一种互补的方法中，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进行鉴定，并在荧光素酶报告翻译试验中进行验证。这些数据将检验一种假设，即细胞内的IRES序列可能不限于5ʹ非翻译区，而是可能在人类基因组中更广泛地发现。总之，这些实验扩展了我们对蛋白质生物合成过程的了解，并为未来在单分子水平上进行蛋白质生物合成的机制研究奠定了基础。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.