BRC-BIO Structural regulation of cap-independent translation in eukaryotic mRNAs.

信使核糖核酸（mRNA）分子携带用于制造蛋白质的遗传密码，可以折叠成复杂的结构。mRNA还可以与控制其功能的蛋白质结合。 在高等生物中，mRNA的翻译需要一系列复杂的、严格调控的起始步骤。然而，在细胞应激（低氧，低营养，DNA损伤）下，当大多数mRNA翻译被阻断时，一些mRNA可以绕过监管检查。这些导致某些蛋白质水平高于正常水平。目前还不清楚相同的mRNA分子在有压力和没有压力的情况下如何不同地运作。该项目旨在通过研究细胞应激条件下mRNA折叠和相互作用的模式来弥合这一关键的知识差距。本科研究人员在格鲁吉亚的指定公立文科学院位于米勒奇维尔，佐治亚州将进行这些调查。学生将利用尖端的生物技术工具，包括下一代测序和计算分析。学生还将从与研究密集型大学的合作者的互动中获益。为了扩大参与该项目的学生人数，部分将作为基于课程的研究经验进行，参加分子生物技术课程的学生将在一个学期内通过动手，脚手架项目进行指导。来自邻近县的高中生将通过细胞和分子生物学研讨会参与进来，以打破未来本科研究人员之间的一些障碍。一年级学生也将直接参与研究的机会，旨在提高保留和学生的成功高影响力的做法。对于细胞mRNA，从标准的帽依赖性翻译转换为应激诱导的帽非依赖性翻译知之甚少。在这样的mRNA的子集中，内部核糖体进入位点（IRES）已经被报道参与RNA结构介导的翻译起始。对于许多报道的细胞IRES没有可靠的结构数据是目前可用的，而且在细胞内条件下的结构的调查是缺乏的。该项目旨在弥合这一关键的知识差距，从人类细胞系中选择一小部分推定的含IRES的mRNA开始。将通过探测应激诱导的人活细胞系中的IRES区域来检测从帽依赖性翻译转换为帽非依赖性翻译的转换机制期间的结构变化。将应用化学探测试剂，如2A 3，NAI和5 NIA，可以可靠地探测活细胞内的RNA结构。此外，将使用交联试剂检测RNA-蛋白质相互作用。将在强制降解条件下比较靶RNA区域上蛋白质相互作用位点的模式。总的来说，这些数据将提供一个机制框架的基础上，在压力下的调节RNA的结构变化。数据验证将采用蛋白质印迹法、使用工程mRNA转录构建体的报告基因测定法和基于CLIP的策略。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Messenger ribonucleic acid (mRNA) molecules carry the genetic code used to make proteins and can fold into intricate structures. mRNAs can also bind to proteins that control their function. The translation of mRNA in higher organisms, requires a series of complex, tightly regulated initiation steps. However, under cellular stress (low oxygen, low nutrients, DNA damage) when most mRNA translation is blocked, some mRNAs can bypass regulatory checks. These leads to elevated levels of certain proteins compared to normal levels. It is unclear how the same mRNA molecule operates differently with and without stress. This project aims to bridge this critical gap in knowledge by investigating the patterns of mRNA folding and interactions under cellular stress conditions. Undergraduate researchers at Georgia’s designated public liberal arts college located in Milledgeville, GA will conduct these investigations. Students will utilize cutting-edge biotechnology tools including next-generation sequencing and computational analysis. Students will also gain from interactions with collaborators at research-intensive universities. To broaden the number of students participating in this project, portions will be conducted as a course-based research experience, where students enrolled in a Molecular BioTechniques course will be guided through hands-on, scaffolded projects over a semester. High school students from neighboring counties will be engaged via workshops in cell and molecular biology to break some barriers among future undergraduate researchers. First-year students will also be directly engaged research opportunities, a high-impact practice that is designed to boost retention and student success. For cellular mRNAs, switching from the standard cap-dependent translation to stress-induced cap-independent translation is poorly understood. In a subset of such mRNAs, internal ribosome entry sites (IRESs) have been reported which engage in RNA-structure mediated initiation of translation. For many reported cellular IRESs no reliable structure data is currently available, and furthermore investigation of structures under in-cell conditions is lacking. This project aims to bridge this critical gap in knowledge starting with a small selection of putative IRES-containing mRNAs from human cell lines. Structural changes during the switching mechanism from cap-dependent to cap-independent translation will be detected by probing IRES-region in stress-induced live human cell lines. Chemical probing reagents such as 2A3, NAI, and 5NIA will be applied that can probe RNA structure reliably within live cells. Furthermore, RNA-protein interactions will be detected using crosslinking reagents. Patterns of protein interaction sites on target RNA regions will be compared under stress conditions. Collectively, this data will provide a mechanistic framework based on structural changes in regulatory RNAs under stress. Data validation will apply western blots, reporter assays using engineered mRNA transcript constructs, and CLIP-based strategies.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.