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

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.