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。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。核糖体不随机与mRNA结合；取而代之的是，有一个高度调节的过程，可以确保核糖体在特定位置与代码结合，以便将mRNA代码正确转换为蛋白质。尽管在大多数细胞mRNA中都有这种倡议发生的标准途径，但在某些条件（例如应力）下，在细胞中存在替代途径。了解这些替代途径也很重要，因为许多病毒都探索它们。该项目研究了一种替代蛋白质生物合成途径，称为内部倡议。内部主动性要求RACK1蛋白与核糖体结合，该项目将研究该蛋白的原因。通过替代途径翻译的mRNA也具有内部启动所需的结构特征。提出的工作将筛选代表数千个细胞mRNA的序列，以识别能够结合含有RACK1的核糖体并允许内部翻译起始的RNA结构。这些方法将大大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。该研究计划是由一项教育生活与学习计划完成的，该计划鼓励我们的本科生群体从事科学，技术，工程或数学的职业。该计划的生物学学生的第一年生活，饮食和一起学习并每周开会，谈论他们过渡到大学的过渡，找到与科学教师在本科生职业早期与科学教师进行研究的机会，以及在科学专业和职业中取得成功的策略。该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RNA结构，绕过了mRNA的M7G帽的要求，以促进翻译。尽管已经对病毒IRESS进行了深入的研究，但细胞IRES序列的理解较少。为了研究细胞IRES介导的翻译的机制，将检查核糖体蛋白RACK1与启动因子EIF3D和DAP5的相互作用，并将确定这些因素通常调节的RNA。在完整的方法中，将通过高通量测序识别在选择后的圆形RNA构建体上启动的RNA序列，并在荧光素酶报道器翻译测定中进行验证。这些数据将检验以下假设：细胞IRES序列可能不限于5ʹ未翻译的区域，但可以在人类基因组中更广泛地发现。这些实验共同扩大了我们对蛋白质生物合成过程的了解，并为在单分子水平上对蛋白质生物合成进行未来的机械研究奠定了基础。这奖反映了NSF的法定任务，并被认为是通过基金会的知识分子和更广泛的影响审查标准来通过评估来通过评估来支持的。

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 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

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