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.

项目摘要 本研究的目的是描述逆转录酶的酶活性 (RT)人长散布元件1（LINE-1，L1 RT）的表达。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%，因为它也 导致短散布元件的增殖（例如，Alu）和其他逆向元素。L1 RT活性也是 作为一种驱动力背后的各种人类疾病，如黄斑变性，黄斑变性， 古铁雷斯综合征和系统性红斑狼疮。LINE-1表达和反转录转座是 常见于许多癌症，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， 称为靶向启动逆转录（TPRT）。L1 RT由第二开放阅读帧编码 L1的ORF 2，存在于被称为ORF 2 p的蛋白质中。在这个提议中，我证明了我已经分离出了ORF 2 p RT结构域，并且这在体外具有RT活性。我打算使用这个蛋白质结构域，以及全长 ORF 2 p和L1 RNP来充分表征L1 RT的生化和酶性质。 具体目标，我将描述其持续性，这是一个指标，是否RT停止和暂停，或 它是否会一直运行直到到达模板的末尾。我还将测量L1 RT的保真度， 定义了它从RNA模板复制DNA的忠实程度。最后，我提出初步测序数据， 显示L1 RT具有在没有引物的情况下开始加工RNA模板的意外能力， 提出了严格的实验来研究这一特征。在我的第二个具体目标中，我专注于当 L1 RT到达其模板的末端，这可能对理解细胞如何修复中间产物至关重要。 换位我目前的初步数据表明，L1 RT增加了额外的核苷酸，其cDNA的末端， 建议开展研究，充分说明这一活动的特点。此外，我将研究L1 RT切换到 一个模板末端的不同模板以及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 改性 这项工作将推进我们对LINE-1在细胞内如何运作以及LINE-1如何在细胞内发挥作用的理解。 完成它的生命周期。这项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DNA损伤剂和模式识别的触发器的信号传导 受体，分别。最后，本研究将深入了解LINE-1类逆行素的演化 存在于所有生命王国中。