Mechanism and Regulation of non-canonical translation initiation

非规范翻译启动机制及调控

• 批准号: 2047629
• 负责人: Gabriele Fuchs
• 金额: $ 59.91万
• 依托单位: SUNY at Albany
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2047629&HistoricalAwards=false
• 关键词: Mechanism; Regulation; non; canonical; translation

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.

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mRNA来源于细胞染色体中的DNA。核糖体不是随机结合mRNA的；相反，有一个高度调控的过程来确保核糖体在特定的地方与编码结合，这样mRNA编码就被正确地翻译成蛋白质。虽然大多数细胞mrna的起始都有一个标准的途径，但在某些条件下（如应激），细胞中也有其他途径。了解这些替代途径也很重要，因为许多病毒会利用它们。本项目研究了一种被称为内部起始的蛋白质生物合成途径。内部起始需要RACK1蛋白与核糖体结合，本项目将研究需要该蛋白的原因。通过其他途径翻译的mRNA也具有内部起始所需的结构特征。这项工作将筛选代表数千种细胞mrna的序列，以鉴定能够结合含有RACK1的核糖体并允许内部翻译起始的RNA结构。这些方法将极大地增强我们对内部起始介导的蛋白质生物合成的成分和机制的理解。这个研究项目是一个教育生活和学习项目的补充，鼓励不同群体的本科生追求科学、技术、工程或数学方面的职业。生物学专业一年级的学生一起生活、吃饭和学习，每周见面讨论他们向大学的过渡，寻找机会在本科生涯早期与科学教师一起做研究，以及在科学专业和职业生涯中取得成功的策略。这个研究项目将有助于培养下一代科学家。内部核糖体进入位点（IRES）是绕过mRNA的m7G帽以促进翻译的RNA结构。尽管病毒IRES已被深入研究，但细胞IRES序列尚不清楚。为了研究ires介导的细胞翻译机制，我们将研究核糖体蛋白RACK1与起始因子eIF3D和DAP5的相互作用，并鉴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结构上启动的RNA序列将通过高通量测序进行鉴定，并在荧光素酶报告基因翻译试验中进行验证。这些数据将验证细胞IRES序列可能不局限于5′非翻译区，而可能更广泛地存在于人类基因组中的假设。总之，这些实验扩展了我们对蛋白质生物合成过程的认识，为今后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。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

The dynamics of subunit rotation in a eukaryotic ribosome.

• DOI: 10.3390/biophysica1020016
• 发表时间: 2021-06
• 期刊: Biophysica

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis.

• DOI: 10.1016/j.devcel.2022.03.005
• 发表时间: 2022-04-11
• 期刊: DEVELOPMENTAL CELL
• 影响因子: 11.8
• 作者: Martin, Elliot T.;Blatt, Patrick;Nguyen, Elaine;Lahr, Roni;Selvam, Sangeetha;Yoon, Hyun Ah M.;Pocchiari, Tyler;Emtenani, Shamsi;Siekhaus, Daria E.;Berman, Andrea;Fuchs, Gabriele;Rangan, Prashanth
• 通讯作者: Rangan, Prashanth

A Split NanoLuc Reporter Quantitatively Measures Circular RNA IRES Translation.

• DOI: 10.3390/genes13020357
• 发表时间: 2022-02-16
• 期刊: Genes
• 影响因子: 3.5
• 作者: Sehta P;Wilhelm AM;Lin SJ;Urman MA;MacNeil HA;Fuchs G
• 通讯作者: Fuchs G