A quick and simplified method (QuickRibo-mRNA) for isolation of ribosome protected mRNA fragments for translatome identification

本研究的目的是建立和验证一种新的核糖体保护的分离方法， 用于翻译组鉴定的mRNA足迹。翻译组或核糖体分析（RP），深度测序 核糖体保护的mRNA足迹技术，是一种强大的方法，可以提供一个“快照”的积极 翻译mRNA。彻底改变在亚密码子处捕获mRNA翻译的每一步的能力 分辨率，它潜在地允许人们监测细胞中mRNA翻译的速率和位置，并注释 基因组的新蛋白质编码序列。因此，RP为探索和建模提供了巨大的机会 真实的时间复杂的细胞过程。这种方法已被广泛挖掘在许多疾病 包括癌症免疫学和神经学RP从COVID中鉴定出几种新的微肽和蛋白质 mRNA，进一步扩大其作为一种新的靶点识别方法的潜力。在此背景下，RP应运而生 作为一种有效的方法来确定mRNA移码和新的ORF，并提供有价值的见解， 了解基因组的功能元件，疾病的发病机制和影响治疗的评价， mRNA治疗和以基因和组织特异性方式的癌症治疗的情况。然而，RP 应用受到阻碍，并限于专业实验室，因为它需要专业知识，专业仪器 而且耗时-平均耗时约5天。反过来，这又会影响最终数据的成本和质量 从而模糊了mRNA的翻译前景。鉴于其广泛使用的能力和潜力， 迫切需要类似于RNA纯化方法的替代/改进的RP技术 其耗时更少、没有伪像并且便宜。在第一阶段的应用中，我们提出了两个具体的 旨在完善QuickRibo-mRNA方法，验证并与当前RP方法进行比较，以建立其 功效在aim-1中，我们将建立并验证QuickRibo-mRNA方法学在各种条件和细胞中的应用。 在获得均匀的核糖体保护的mRNA片段中具有改进的读段映射到编码区的百分比 序列的在aim-2中，我们将比较QuickRibo-mRNA与已知核糖体分析的功效。 技术.我们认为，第一阶段的建议将为第二阶段提供一个基准， QuickRibo-mRNA方法用于进行核糖体分析，具有高重现性和高效率。最后 RP将以最简单的方法熟练地完成，只需较少的时间，仪器和专业知识来鉴定基因 “表达与阅读框架和翻译效率”之前作出任何结论完全基于 RNA-基因“表达谱”。

The goal of this proposal is to establish and validate a novel method for the isolation of ribosome protected mRNA footprints for translatome identification. Translatome or Ribosome profiling (RP), a deep sequencing technique of ribosome-protected mRNA footprints, is a powerful method that gives a “snapshot” of actively translating mRNA. Revolutionizing the capacity to capture every step of mRNA translation at sub-codon resolution, it potentially allows one to monitor the rate and location of mRNA translation in the cell and annotate the new protein coding sequences of genomes. RP, therefore, affords a vast opportunity to explore and model complex cellular processes in real time. This methodology has been mined extensively in numerous diseases including cancer, immunology and neurology. RP identified several new micro-peptides and protein from COVID mRNA, further expanding its potential as a novel target-identification method. In this context, RP has emerged as a potent method to identify mRNA frameshifting and novel ORFs, and providing valuable insights for understanding functional elements of the genome, disease pathogenesis and evaluation of impact treatment in the case of mRNA therapeutics and cancer therapy in a gene and the tissues specific manner. However, RP applications are stunted and limited to specialized labs because it requires expertise, specialized instruments and time-consuming - taking up to ~5 days on an average. In turn, this affects the cost, quality of the final data and thereby obscuring the mRNA translational landscape. Given the power and potential for widespread use, there is an urgent and unmet need for alternative/improved RP technology similar to RNA purification methods which are less time consuming, free of artifacts and inexpensive. In Phase-I application, we proposed two specific aims to refine the QuickRibo-mRNA method, validate and compare it with current RP methods to establish its efficacy. In aim-1 we will establish and validate the QuickRibo-mRNA methodology in various conditions and cell types in obtaining uniform ribosome protected mRNA fragments with improved percent read mapping to coding sequences. In aim-2 we will compare the efficacy of QuickRibo-mRNA with known ribosome profiling technologies. We believe that phase-I proposal will provide a benchmark for phase II, which can streamline the QuickRibo-mRNA method for performing ribosome profiling with high reproducibility and efficiency. In the end RP will be done proficiently in the simplest way with less time, instrumentation and expertise to identify gene “expression with reading frames and translation efficiency” before making any conclusions entirely based on RNA-gene “expression profile”.