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

项目总结 G-四链体(G4)是一种非规范的二级结构，形成于DNA和RNA的富含鸟嘌呤片段中。 生物信息学揭示了超过375,000个潜在的G4形成序列(PQ)分布在整个人类 基因组。当转录时，这样的PQS可以产生两种结构，mRNA中的RNAG4(RG4)和R-loop，以及 RNA：由新生的富含G的RNA和富含C的模板DNA退火而形成的DNA杂交结构。近期 研究发现，R环结构与PQS重叠，表明每个元素发挥的作用不同。 RG4在包括关键癌基因在内的许多基因的5‘UTR区中密度最高，而R-环则丰富 在启动子区域附近。尽管越来越多的证据表明RG4和R环都是 在基因调控方面，目前仍不确定它们如何调控转录和翻译。此外，无论是RG4 而R-环结构可以被特定靶向它们的蛋白质调节，这增加了复杂性 它们在基因调控中的影响。重要的是，这些特定的蛋白质是癌症的标志，强调了 它们在肿瘤发生中扮演的角色。我建议在体外和基于细胞的系统中研究RG4和R-loop 解决以下问题：1)PQS序列的组成和位置是如何在5‘非编码区编码的 DNA片段调控转录和翻译？2)R-loop和RnaseH1(R- 3)RG4和DHX36(RG4)在转录和翻译中的作用 在转录和翻译中分解解旋酶)？我将使用单分子技术的组合(单分子 分子FRET、单分子PIFE和超分辨率细胞成像)和集成分析(双荧光素酶 分析、整体转录分析、实时G4形成测量、EMSA和核糖体分析)以 实现所提出的目标。我提议的研究得到了与G4领域专家的合作支持 介导的细胞过程(帕特里夏·奥普雷斯科)，G4解旋酶(禤浩焯·费尔-达马雷)，超分辨率 显微镜(Taekjip Ha)和翻译(Rachel Green)(见支持函)。专业发展 机会加上强有力的合作和指导，将使我成为一名富有成效的校长 未来的调查员。我的计划是成为单分子技术方面的专家，除了磨练我的 科学的写作和沟通能力将使我在其他寻求有竞争力的博士后中脱颖而出 各就各位。

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.