Characterization of LINE-1 reverse transcriptase activity

LINE-1 逆转录酶活性的表征

• 批准号: 10604881
• 负责人: Benedict Smail
• 金额: $ 2.29万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-02 至 2023-05-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10604881
• 关键词: Aging; Amino Acids; Autoimmune Diseases; Biochemical; Biological Assay; Bombyx mori; Cancer Biology; Cells; Chromosomal Rearrangement; Clinical; Code; Colon Carcinoma; Cytoplasm; DNA; DNA Damage; DNA Nucleotidylexotransferase; DNA Repair; DNA Sequence Rearrangement; Data; Degenerative Disorder; Disease; Dissociation; Evolution; Gel; Genetic Transcription; Genome; Genomics; Goals; Human; Human Genome; Hydrogen Bonding; Immune signaling; In Vitro; Interferons; Introns; L1 Elements; Label; Length; Lesion; Life; Life Cycle Stages; Long Interspersed Elements; Macular degeneration; Malignant Neoplasms; Malignant neoplasm of esophagus; Measures; Mediating; Modification; Nucleic Acids; Nucleosides; Nucleotides; Open Reading Frames; Pattern recognition receptor; Phenotype; Phylogenetic Analysis; Polymerase; Polymers; Process; Proliferating; Property; Proteins; Pseudouridine; RNA; RNA Binding; RNA Caps; RNA Polymerase II; RNA Processing; RNA vaccine; RNA-Binding Proteins; RNA-Directed DNA Polymerase; Reaction; Reading; Research; Research Proposals; Resolution; Retroelements; Retrotransposition; Retrotransposon; Reverse Transcriptase Inhibitors; Reverse Transcription; Ribonucleotides; Role; Series; Short Interspersed Nucleotide Elements; Signal Transduction; Structure; Systemic Lupus Erythematosus; Tertiary Protein Structure; Testing; Thumb structure; Transcript; Work; clinically significant; driving force; experimental study; human disease; improved; in vitro activity; insight; interest; knowledge base; malignant breast neoplasm; polymerization; pseudotoxoplasmosis syndrome; public health research; repaired

Project Summary The objective of this research proposal is to characterize the enzymatic activities of the reverse transcriptase (RT) of the human long interspersed element 1 (LINE-1, L1 RT). LINE-1 sequences constitute 17% of the human genome, and L1 RT activity is responsible for approximately 40% of our genome because it has also led to the proliferation of short interspersed elements (e.g., Alu), and other retroelements. L1 RT activity is also implicated as a driving force behind a variety of human diseases such as macular degeneration, Aicardi- Goutières syndrome, and systemic lupus erythematosus. LINE-1 expression and retrotransposition are commonplace in numerous cancers, including 90-100% of breast, colon, and esophageal cancers, making understanding the basic biochemical activity of L1 RT relevant to public health research. Reverse transcription is key to the ability of LINE-1 to self-propagate in our genome, through a process known as target-primed reverse transcription (TPRT). L1 RT is encoded by the second open reading frame (ORF2) of L1, residing in the protein known as ORF2p. In this proposal, I show that I have isolated the ORF2p RT domain, and that this has RT activity in vitro. I intend to use this protein domain, as well as full-length ORF2p, and L1 RNPs to fully characterize the biochemical and enzymatic properties of L1 RT. In my first Specific Aim, I will characterize its processivity, which is an indication of whether the RT stops and pauses, or whether it keeps going until it reaches the end of a template. I will also measure the fidelity of L1 RT, which defines how faithfully it copies DNA from an RNA template. Finally, I present preliminary sequencing data showing that L1 RT has the unexpected ability to begin processing an RNA template without a primer and propose rigorous experiments to study this feature. In my second Specific Aim, I focus on what happens when L1 RT reaches the end of its template, which may be crucial to understanding how cells repair intermediates of transposition. I present preliminary data showing that L1 RT adds extra nucleotides to the end of its cDNA and propose studies to fully characterize this activity. Additionally, I will study the ability of L1 RT to switch to a different template at the end of one template and whether LINE-1 RT can use modified RNA as a template, such as the pseudouridine found in mRNA vaccines or the naturally occurring N6-methyladenosine modification. This work will advance our understanding of how LINE-1 operates inside cells and how LINE-1 completes its life cycle. The proposed research will have implications for cancer biology and innate immune signaling where L1 RT is increasingly recognized as a DNA damaging agent and trigger for pattern recognition receptors, respectively. Finally, this research will give insight into the evolution of LINE-1-like retroelements that are found across all kingdoms of life.

项目概要 本研究计划的目的是表征逆转录酶的酶活性 (RT) 人类长散布元件 1 (LINE-1, L1 RT)。 LINE-1 序列占 17% 人类基因组，L1 RT 活性负责我们基因组的大约 40%，因为它还 导致短散布元件（例如 Alu）和其他逆转录元件的增殖。 L1 RT 活动也 被认为是多种人类疾病的驱动力，例如黄斑变性、心脏病 古蒂埃综合征和系统性红斑狼疮。 LINE-1 表达和逆转录转座是 在多种癌症中很常见，包括 90-100% 的乳腺癌、结肠癌和食道癌， 了解与公共卫生研究相关的 L1 RT 的基本生化活性。 逆转录是 LINE-1 在我们的基因组中自我繁殖能力的关键，通过一个过程 称为靶标引发的逆转录（TPRT）。 L1 RT 由第二个开放阅读框编码 L1 的 ORF2（ORF2），位于称为 ORF2p 的蛋白质中。在这个提案中，我表明我已经隔离了 ORF2p RT 结构域，且其在体外具有 RT 活性。我打算使用这个蛋白质结构域以及全长 ORF2p 和 L1 RNP 可全面表征 L1 RT 的生化和酶学特性。在我的第一个 具体目标，我将描述其持续性，这表明 RT 是否停止和暂停，或者 是否继续运行直到到达模板末尾。我还将测量 L1 RT 的保真度， 定义它从 RNA 模板复制 DNA 的忠实程度。最后，我给出初步的测序数据 表明 L1 RT 具有意想不到的能力，可以在没有引物的情况下开始处理 RNA 模板，并且 提出严格的实验来研究这个特性。在我的第二个具体目标中，我关注的是当 L1 RT 到达其模板的末端，这对于理解细胞如何修复中间体可能至关重要 换位。我提供的初步数据表明，L1 RT 在其 cDNA 末端添加了额外的核苷酸，并且 提出研究以充分描述这项活动的特征。此外，我将研究 L1 RT 切换到 一个模板末尾的不同模板以及LINE-1 RT是否可以使用修饰的RNA作为模板， 例如 mRNA 疫苗中发现的假尿苷或天然存在的 N6-甲基腺苷 修改。 这项工作将加深我们对 LINE-1 在细胞内如何运作以及 LINE-1 如何运作的理解 完成其生命周期。拟议的研究将对癌症生物学和先天免疫产生影响 L1 RT 越来越多地被认为是一种 DNA 损伤剂和模式识别触发器 分别是受体。最后，这项研究将深入了解 LINE-1 样逆转录元件的进化 在生命的所有王国中都能找到它们。