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

项目摘要 G-四链体（G4）是一种非典型的二级结构，形成于富含鸟嘌呤的DNA和RNA片段中。 生物信息学揭示了超过375，000个潜在的G4形成序列（PQS）位于整个人类。 基因组当转录时，这种PQS可以产生两种结构，mRNA和R环中的RNA G4（RG 4）， RNA：由新生的富含G的RNA和富含C的模板DNA退火产生的DNA杂交结构。最近 研究表明，R-环结构与PQS重叠，表明每个元件所起的作用不同。 RG 4在许多基因（包括关键癌基因）的5 'UTR中最密集，而R环富集 在启动子区域附近。尽管越来越多的证据表明RG 4和R环都是导致糖尿病的重要因素， 基因调控，它仍然不确定它们如何调节转录和翻译。此外，RG 4 而R环结构可以被专门针对它们的蛋白质调节，这增加了复杂性。 它们对基因调控的影响。重要的是，这些特定的蛋白质是癌症的标志，强调了癌症的特征。 在肿瘤发生中的作用。我建议在体外和基于细胞的系统中研究RG 4和R环， 解决了以下问题：1）PQS序列在5 'UTR中的组成和编码位置如何 DNA片段调控转录和翻译？2)R-loop和RnaseH 1（R- 环消化酶）在转录和翻译中的作用？3)RG 4和DHX 36（RG 4）的贡献是什么 解旋酶）在转录和翻译中的作用？我将使用单分子技术的组合（单 分子FRET、单分子PIFE和超分辨率细胞成像）和系综测定（双荧光素酶 分析、系综转录分析、实时G4形成测量、EMSA和核糖体分析）， 实现提出的目标。我提出的研究得到了与G4领域专家合作的支持 介导的细胞过程（Patricia Opresko），G4解旋酶（Adrian Ferre-D 'Amare），超分辨率 显微镜（Taekjip Ha）和翻译（Rachel绿色）（参见支持信）。专业发展 沿着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。我的计划是成为一个单分子技术专家，除了磨练我的 科学写作和沟通技巧将使我从其他寻求有竞争力的博士后中脱颖而出 岗位

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.