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

迈向基因组工程：位点特异性靶向非 LTR 逆转录元件的 RNA 结合和 cDNA 合成的精确原理

• 批准号: 10377343
• 负责人: Briana Nicole Van Treeck
• 金额: $ 6.98万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377343
• 关键词: 3' Untranslated Regions; Address; Affinity; Amino Acids; Architecture; Binding; Biochemical; Biological Assay; Biology; Bypass; Cell physiology; Complementary DNA; Complex; DNA; DNA Double Strand Break; DNA Transposable Elements; Development; Elements; Engineering; Eukaryota; Evolution; Exhibits; Foundations; Future; Gene Expression; Genes; Genome; Genome engineering; Goals; Human Genome; Incentives; Investigation; Length; Life; Mediating; Methods; Modeling; Molecular; Phylogenetic Analysis; Process; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Directed DNA Polymerase; RNA-Protein Interaction; RRM2 gene; Research; Resources; Retroelements; Retrotransposon; Reverse Transcription; Ribonucleoproteins; Ribosomal DNA; Selfish Genes; Site; Specificity; Structure; Study models; Tertiary Protein Structure; Testing; Training; Transcript; Transgenes; Work; cancer genome; career; design; gene therapy; homologous recombination; insight; personalized health care; personalized medicine; preservation; prospective; repaired; therapeutic gene; therapy development; tool; tumor progression

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.

摘要 转座因子是自私的基因，广泛存在于生命的所有领域，能够移动到 基因组中的新位点。非长末端重复序列（non-LTR）逆转录元件，最古老和最多样化的 真核逆转录转座子和人类基因组中最普遍和最活跃的转座因子， 涉及许多宿主细胞过程，包括发育、基因表达、癌症进展 和基因组进化。非LTR的追溯元素通过一个称为靶启动逆转的过程整合 逆转录转录（TPRT），其中逆转录元件蛋白将逆转录元件RNA直接逆转录成逆转录蛋白。 基因组这种机制绕过了双链DNA断裂的产生，并避免了对双链DNA断裂的需要。 同源重组，为彻底理解蛋白质和RNA特征提供了动力， 这一过程对于未来基因治疗的努力。位点特异性R2非LTR逆向元件是主要的 研究TPRT的模型。尽管蛋白质和模板发生了变化，但R2迁移率的进化持续性 RNA序列提供了阐明基本要求以及系统发育的机会 贸易政策审查小组机制的多样化。这项工作提出了生物化学、分子和 细胞方法来阐明R2逆转录元件蛋白和RNA之间的亲和力的原理， 破译其相互作用的非常高的特异性的原则，并实现完整的TPRT介导的 转基因插入这个项目将建立在我在RNA生物学的基础上，并推动我的研究事业 因为我接受了凯瑟琳柯林斯博士的严格训练，她在RNA-蛋白质相互作用方面有专长， 核糖核蛋白复合物形成和逆转录酶。这项工作将有助于更大的 了解进化如何磨练和多样化的反向元件蛋白质的成对特异性， 除了进一步实现利用非LTR反转录转座子进行转基因导入的最终目标之外，RNA也是一个重要的研究领域。