Towards Genome Engineering: Principles of precision in RNA-binding and cDNA-synthesis for a site-specifically targeted non-LTR retroelement

摘要 转座元件是一种自私的基因，广泛存在于生命的各个领域，能够转移到 基因组中的新位置。非长末端重复序列(Non-LTR)逆转录元件，是祖先中最多样化的 真核反转录转座子和人类基因组中最普遍和最活跃的转座元件， 与许多宿主细胞过程有关，包括发育、基因表达、癌症进展 和基因组进化。非LTR逆转录元件通过一种称为靶标启动反向的过程进行整合 逆转录(TPRT)，逆转录因子蛋白将逆转录因子RNA直接逆转录为 基因组。这种机制绕过了双链DNA断裂的产生，并避免了 同源重组，为彻底了解蛋白质和RNA功能提供了动力，从而促进 这一过程为未来的基因治疗努力奠定了基础。特定部位的R2非LtR反转录元件一直是主要的 研究TPRT的模型。R2移动性的进化持久性，尽管蛋白质和模板发生了变化 RNA序列，提供了一个阐明基本要求以及系统发育的机会 多样化，TPRT机制。这项工作提出了生化、分子和 用细胞方法阐明R2逆转录元件蛋白和RNA之间的亲和力原理，以 破译它们相互作用的高度特异性的原理，并实现完全的TPRT中介 转基因植入。这个项目将建立在我在RNA生物学方面的基础上，并推动我的研究事业 我在凯瑟琳·柯林斯博士的指导下接受了严格的培训，她在RNA-蛋白质相互作用方面拥有专业知识， 核糖核蛋白复合体的形成和逆转录酶。这项工作将为更大的 了解进化如何磨练和多样化逆转录元件蛋白的成对特异性 除了进一步利用非LTR反转录转座子进行转基因导入这一最终目标之外，还可以利用RNA.

ABSTRACT Transposable elements are selfish genes widespread in all domains of life that are capable of moving to new sites in the genome. Non-long terminal repeat (non-LTR) retroelements, the ancestral and most diverse eukaryotic retrotransposons and the most prevalent and active transposable elements in the human genome, are implicated in a number of host cell processes including development, gene expression, cancer progression and genome evolution. Non-LTR retroelements integrate through a process called target-primed reverse transcription (TPRT) in which the retroelement protein reverse transcribes the retroelement RNA directly into the genome. This mechanism bypasses the creation of a double-stranded DNA break and avoids the need for homologous recombination, providing incentive to thoroughly understand protein and RNA features that facilitate this process for future gene therapy endeavors. The site-specific R2 non-LTR retroelement has been the primary model for studying TPRT. The evolutionary persistence of R2 mobility, despite changes in protein and template RNA sequence, provides an opportunity to elucidate fundamental requirements for, as well as phylogenetic diversification of, the TPRT mechanism. This work proposes a combination of biochemical, molecular and cellular approaches to elucidate the principles of the affinity between the R2 retroelement protein and RNA, to decipher the principles of the very high specificity of their interaction and to achieve complete TPRT-mediated transgene insertion. This project will build upon my foundation in RNA biology and advance my research career as I receive rigorous training under Dr. Kathleen Collins, who has expertise in RNA-protein interaction, ribonucleoprotein complex formation, and reverse transcriptases. This work will contribute to a greater understanding of how evolution has honed and diversified the pairwise specificity of a retroelement protein and RNA in addition to furthering the eventual goal of exploiting non-LTR retrotransposons for transgene introduction.