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

信使核糖核酸(信使核糖核酸)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。MRNAs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翻译被阻止时，一些mRNAs可以绕过监管检查。这会导致某些蛋白质水平高于正常水平。目前还不清楚在有压力和没有压力的情况下，同一个mRNA分子的运作方式是如何不同的。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鸿沟。位于佐治亚州米尔奇维尔的佐治亚州指定公立文科学院的本科生研究人员将进行这些调查。学生将利用尖端生物技术工具，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受益。为了扩大参与这个项目的学生的数量，部分将作为基于课程的研究体验进行，在这个过程中，注册分子生物技术课程的学生将在一个学期的时间里通过动手的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，以打破未来本科生研究人员的一些障碍。一年级学生还将获得直接参与研究的机会，这是一种旨在提高留校率和学生成功程度的高影响力实践。对于细胞内的mRNAs，从标准的帽依赖翻译转换到应力诱导的帽非依赖性翻译还知之甚少。在这类mRNAs的一个子集中，已经报道了参与RNA结构介导的翻译起始的内部核糖体进入位点(IRESS)。对于许多已报道的细胞IRESS，目前还没有可靠的结构数据，而且还缺乏对细胞内条件下的结构的研究。该项目旨在弥合这一关键的知识差距，首先从人类细胞系中挑选出一小部分可能含有IRES的mRNAs。通过探测应激诱导的活细胞系中的IRES区域，可以检测到从帽依赖到帽非依赖翻译机制的结构变化。将应用2A3、NAI和5NIA等化学探测剂，可以可靠地探测活细胞内的RNA结构。此外，将使用交联剂检测RNA与蛋白质的相互作用。在胁迫条件下，将比较目标RNA区域上蛋白质相互作用位点的模式。总而言之，这些数据将提供一个基于压力下调控RNA结构变化的机制框架。数据验证将应用Western blotts、使用工程mRNA转录结构的记者分析和基于片段的策略。该奖项反映了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。

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.