EAGER: Measuring transcriptome-wide translation initiation rates from a single experiment

翻译是核糖体读取并使用mRNAs中的遗传信息来产生具有特定细胞功能的蛋白质的过程。翻译起始率是翻译输出的关键指标，因为这一速率规定了每个mRNA可以产生多少蛋白质，从而决定了细胞中可以获得多少特定蛋白质。这个项目的目的是检查和优化一种在全基因组水平上确定翻译起始率的新方法。这种方法将允许在单个实验中快速有效地确定任何类型细胞的转录组范围的翻译起始率。最终，这项技术将使研究人员能够更广泛和更深入地解决有关翻译过程的基本问题。理论实验室和实验实验室小组之间的工作人员交流将使研究生和博士后学者学习互补的实验和理论技能，这将加强他们作为定量生物学家和生物物理学家的培训。核糖体图谱被广泛用于获得体内翻译的高分辨率图谱。通常，核糖体图谱实验与RNA测序实验在同一样本上并行进行。然而，由于文库生成方面的巨大技术差异，以及这两个独立实验之间缺乏允许标准化的绝对标准，所产生的核糖体图谱和RNA测序数据在数量上不具有可比性。这种传统的方法还使所需的测序量翻了一番，并在比较产生的DNA文库时引入了额外的统计不确定性。在这项急切的提案中，研究人员的理论和实验专业知识将结合起来，创建一种新的实验方案，以定量测量单个实验的信使RNA丰度和翻译，从而量化体内整个转录组的绝对翻译起始率。这一新的实验方案将是核糖体图谱的变体，将RNA测序和核糖体图谱的特征结合到一个实验方法中。这消除了在比较核糖体图谱和RNA测序的独立实验时遇到的技术差异，从而可以直接量化整个转录组的翻译起始率。这项技术将超越传统的翻译控制定性指标，如“翻译效率”，并测量活体中蛋白质生产的定量比率。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。

Translation is the process by which ribosomes read and use the genetic information in mRNAs to generate proteins with specific cellular functions. The translation initiation rate is the key measure of translational output, as this rate specifies how much protein can be made from each mRNA and hence how much of a particular protein will be available in a cell. The aim of this project is to examine and optimize a novel approach for determining translation initiation rates at a genome-wide level. This approach will allow transcriptome-wide translation initiation rates to be determined quickly and efficiently in a single experiment and for any type of cell. Ultimately, this technique will allow researchers to address fundamental questions about the process of translation more broadly and deeply. The staff exchanges between the theoretical and experimental laboratory groups will allow graduate students and post-doctoral scholars to learn complementary experimental and theoretical skills that will enhance their training as quantitative biologists and biophysicists.Ribosome profiling is widely used to obtain high-resolution profiles of in vivo translation. Typically, ribosome profiling experiments are carried out in parallel with RNA sequencing experiments on the same sample. The resulting ribosome profiling and RNA sequencing data, however, are not quantitatively comparable due to substantial technical differences in library generation and the lack of an absolute standard permitting normalization between these two independent experiments. This conventional approach also doubles the amount of sequencing required and introduces additional statistical uncertainty in comparing the generated DNA libraries. In this EAGER proposal, the theoretical and experimental expertise of the investigators will be combined to create a new experimental protocol to quantitatively measure messenger RNA abundance and translation from a single experiment, and thereby quantify absolute transcriptome-wide in vivo translation initiation rates. This new experimental protocol will be a variant of ribosome profiling by combining features of RNA sequencing and ribosome profiling into one experimental method. This eliminates the technical differences encountered in comparing the independent experiments of ribosome profiling and RNA sequencing thus allowing direct quantification of transcriptome-wide translation initiation rates. This technique will go beyond conventional qualitative measures of translation control, such as "translation efficiency", and measure quantitative rates of protein production in vivo.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.