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

生物体的许多重要特征，包括其最终大小、对疾病或不利环境因素的抵抗力等，可以追溯到特定基因组的活动水平。因此，了解是什么决定了不同个体的基因活性水平，对于开发即使在不利条件下也能保持高产的更好作物品种至关重要。了解是什么影响基因活性的第一步是开发监测这种活性的方法。由于每个生物体都有数千个基因，因此开发能够同时精确测量数千个基因活性的技术非常重要。传统上，通过监测转录来测量基因的活性，即将DNA序列中包含的遗传指令转化为基于蛋白质的细胞机器的多步骤过程中的第一步。然而，由于基因活性在DNA-蛋白质信息转换过程的后续步骤中也受到调节，因此还需要测量这些下游步骤的活性，因为它们可以强烈影响由特定基因产生的蛋白质的量。该项目的目标是开发一种新的生物技术来量化这一过程的最后一步，即信使RNA中所含信息转化为蛋白质。这项技术将通过降低当前技术的成本和时间要求，并允许发现各种农业重要性状背后的基因调控机制，来支持生物经济。 该提案的主要目标是开发一种高效，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速率。如果成功，RiboPi将使翻译调控信息像RNA-seq用于转录组学一样容易获得，从而降低成本和时间要求、实验程序的复杂性以及所需的生物材料量。这不仅将使翻译分析成为许多实验室的常规技术，而且还可以绕过当前技术的一些限制-例如将非常短的核糖体足迹映射到特定的剪接变体，等位基因甚至多倍体物种中的同源物的困难-或者使一组基因的靶向研究成为可能。为了实现这一目标，我们建议开发RiboPi，这是一种实验上简单的方法，可以捕获每个转录本中的第一个或最后一个核糖体，以及比较不同实验条件下这些核糖体位置分布的计算方法。拟议的实验管道涉及测试体内和体外分子生物学程序的新组合，以有效和特异性地映射转录物中的第一个/最后一个核糖体。使这一提议具有高风险的一些未知因素是：（1）是否能找到合适的实验条件的不确定性（例如，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。

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.