Defining the Role of 5'UTR G-Quadruplex in Transcription and Translation

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PROJECT SUMMARY G-quadruplex (G4) is a non-canonical secondary structure that forms in guanine rich segments of DNA and RNA. Bioinformatics revealed over 375,000 potential G4 forming sequences (PQS) located throughout the human genome. When transcribed, such PQS can give rise to two structures, RNA G4 (RG4) in mRNA and R-loop, an RNA:DNA hybrid structure that results from annealing of nascent G-rich RNA and C-rich template DNA. Recent study revealed that R-loop structures overlap with PQS, suggesting a divergent role played by each element. RG4s are found most densely in the 5’UTR of many genes, including key oncogenes, while R-loops are enriched near the promoter region. Despite the mounting evidence that both RG4s and R-loops are important factors in gene regulation, it remains uncertain how they regulate transcription and translation. Furthermore, both the RG4 and R-loop structures can be modulated by proteins that specifically target them, which adds to the complexity of their impact in gene regulation. Importantly, these specific proteins are hallmarks of cancer, emphasizing the role they play in oncogenesis. I propose to investigate RG4s and R-loop in vitro and in cell-based systems to address the following questions: 1) How does the PQS sequence composition and position encoded in 5’UTR segment of DNA regulate transcription and translation? 2) What is the contribution of R-loop and RnaseH1 (R- loop digesting enzyme) in transcription and translation? 3) What is the contribution of RG4 and DHX36 (RG4 resolving helicase) in transcription and translation? I will use a combination of single molecule techniques (single molecule FRET, single molecule PIFE, and super resolution cell imaging) and ensemble assays (dual-luciferase assays, ensemble transcription assays, real-time G4 formation measurement, EMSA, and ribosome profiling) to achieve the proposed aims. My proposed research is bolstered by collaborations with experts in the fields of G4 mediated cellular processes (Patricia Opresko), G4 helicases (Adrian Ferre-D’Amare), super resolution microscopy (Taekjip Ha), and translation (Rachel Green) (see support letters). Professional development opportunities along with strong collaboration and mentorship will enable me to become a productive principal investigator in the future. My plan to become an expert in single molecule techniques in addition to honing my scientific writing and communication skills will set me apart from others seeking competitive postdoctoral positions.