EAGER: TRTech-PGR: New methods to study gene-specific translation regulation

EAGER：TRTech-PGR：研究基因特异性翻译调控的新方法

• 批准号: 2327912
• 负责人: Jose Alonso
• 金额: $ 30万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2327912&HistoricalAwards=false
• 关键词: EAGER; TRTech; PGR; New; methods

Many important characteristics of an organism, including their final size, resistance to diseases or adverse environmental factors, etc., can be traced back to the levels of activity of specific sets of genes. Understanding what determines these levels of gene activity in different individuals is, therefore, critical to, for example, developing better varieties of crops that can maintain high yields even under adverse conditions. The first step towards understanding what affects the activity of a gene is developing methods of monitoring such activity. Since each individual organism has thousands of genes, it is important to develop technologies that allow the accurate measurement of the activity of thousands of genes in parallel. Traditionally, the activity of a gene is measured by monitoring transcription, i.e. the first step in the multistep process of converting the genetic instructions contained in the DNA sequence into protein-based cellular machines. However, because gene activity is also regulated at later steps in this DNA-to-protein information conversion process, it is also desirable to measure the activity of these downstream steps, as they can strongly affect the amount of proteins produced from a particular gene. The goal of this project is to develop a new biotechnology to quantify the last step of this process, the translation of the information contained in the messenger RNA into proteins. This technology will support the bioeconomy by reducing the cost and time requirements of current technologies and allowing for the discovery of the gene regulation mechanism behind a wide variety of agriculturally important traits. The main objective of this proposal is to develop an efficient, simple, and scalable RiboPi technology to quantify translation rates at both genome-wide and single-gene levels. If successful, RiboPi will make translation regulation information as accessible as RNA-seq did for transcriptomics, reducing the cost and time requirements, the complexity of the experimental procedures, and the amount of biological material needed. Not only will this make translation analysis a routine technique in many labs, but it could also bypass some of the limitations of the current technologies--such as the difficulty of mapping the very short ribosome footprints to specific splice variants, alleles, or even homologs in polyploid species--or enable targeted studies for a group of genes. To achieve this goal, we propose to develop RiboPi, an experimentally simple approach to capture the first or last ribosome in each transcript and the computational methods to compare the distribution of these ribosome positions between different experimental conditions. The proposed experimental pipeline involves testing novel combinations of in vivo and in vitro molecular biology procedures to efficiently and specifically map the first/last ribosome in a transcript. Some of the unknowns that make this proposal high-risk are (1) the uncertainty of whether suitable experimental conditions can be found (e.g., that preserve ribosome binding and promote reverse transcriptase activity but melt the secondary structure of mRNA) and (2) the ability to infer the efficiency of translation from the distributions of first/last ribosomes on transcripts.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.

有机体的许多重要特征，包括其最终大小，对疾病的抵抗力或不利的环境因素等，可以追溯到特定基因集的活动水平。因此，了解不同个体中这些基因活性水平的原因对于例如开发出更好的农作物，即使在不利条件下也可以保持高产量的品种至关重要。了解影响基因活性的第一步是开发监测这种活动的方法。由于每个生物体都有成千上万的基因，因此必须开发允许并联数千个基因活性的技术很重要。传统上，基因的活性是通过监测转录来测量的，即将DNA序列中包含的遗传指令转换为基于蛋白质的细胞机的多步骤过程中的第一步。但是，由于基因活性在此DNA到蛋白信息转化过程的后期步骤中也受到调节​​，因此也需要测量这些下游步骤的活性，因为它们可以强烈影响从特定基因产生的蛋白质的量。该项目的目的是开发一种新的生物技术来量化此过程的最后一步，即信使RNA中包含的信息转换为蛋白质。该技术将通过减少当前技术的成本和时间要求，并允许发现各种重要农业特征背后的基因调节机制来支持生物经济。 该提案的主要目的是开发一种有效，简单和可扩展的Ribopi技术，以量化全基因组和单基因水平的翻译率。如果成功的话，Ribopi将提供像RNA-Seq一样可访问的翻译调节信息，用于转录组学，减少成本和时间要求，实验程序的复杂性以及所需的生物材料量。这不仅会使翻译分析成为许多实验室中的常规技术，而且还可以绕过当前技术的某些局限性（例如，将非常短的核糖体足迹映射到特定的剪接变体，等位基因，甚至是多倍型物种中的困难 - 或对一组基因的靶向研究中的靶向研究。为了实现这一目标，我们建议开发Ribopi，这是一种实验简单的方法，可以捕获每个转录本中的第一个或最后一个核糖体以及计算方法，以比较不同实验条件之间这些核糖体位置的分布。提出的实验管道涉及测试体内和体外分子生物学程序的新型组合，以有效，特异性地绘制转录本中的第一个/最后一个核糖体。 （1）是否可以找到合适的实验条件的一些未知数是（1）是否可以找到合适的实验条件（例如，保留核糖体结合并促进逆转录酶活性，但融化了mRNA的二级结构，但融化了mRNA的次要结构）和（2）从对dectription的第一个/最后的核能分布中推断出逐步划分的分布的能力。使用基金会的智力优点和更广泛的影响评估标准进行评估。