Characterization of LINE-1 reverse transcriptase activity
LINE-1 逆转录酶活性的表征
基本信息
- 批准号:10604881
- 负责人:
- 金额:$ 2.29万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AgingAmino AcidsAutoimmune DiseasesBiochemicalBiological AssayBombyx moriCancer BiologyCellsChromosomal RearrangementClinicalCodeColon CarcinomaCytoplasmDNADNA DamageDNA NucleotidylexotransferaseDNA RepairDNA Sequence RearrangementDataDegenerative DisorderDiseaseDissociationEvolutionGelGenetic TranscriptionGenomeGenomicsGoalsHumanHuman GenomeHydrogen BondingImmune signalingIn VitroInterferonsIntronsL1 ElementsLabelLengthLesionLifeLife Cycle StagesLong Interspersed ElementsMacular degenerationMalignant NeoplasmsMalignant neoplasm of esophagusMeasuresMediatingModificationNucleic AcidsNucleosidesNucleotidesOpen Reading FramesPattern recognition receptorPhenotypePhylogenetic AnalysisPolymerasePolymersProcessProliferatingPropertyProteinsPseudouridineRNARNA BindingRNA CapsRNA Polymerase IIRNA ProcessingRNA vaccineRNA-Binding ProteinsRNA-Directed DNA PolymeraseReactionReadingResearchResearch ProposalsResolutionRetroelementsRetrotranspositionRetrotransposonReverse Transcriptase InhibitorsReverse TranscriptionRibonucleotidesRoleSeriesShort Interspersed Nucleotide ElementsSignal TransductionStructureSystemic Lupus ErythematosusTertiary Protein StructureTestingThumb structureTranscriptWorkclinically significantdriving forceexperimental studyhuman diseaseimprovedin vitro activityinsightinterestknowledge basemalignant breast neoplasmpolymerizationpseudotoxoplasmosis syndromepublic health researchrepaired
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Benedict Smail其他文献
Benedict Smail的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
相似海外基金
Double Incorporation of Non-Canonical Amino Acids in an Animal and its Application for Precise and Independent Optical Control of Two Target Genes
动物体内非规范氨基酸的双重掺入及其在两个靶基因精确独立光学控制中的应用
- 批准号:
BB/Y006380/1 - 财政年份:2024
- 资助金额:
$ 2.29万 - 项目类别:
Research Grant
Quantifying L-amino acids in Ryugu to constrain the source of L-amino acids in life on Earth
量化 Ryugu 中的 L-氨基酸以限制地球生命中 L-氨基酸的来源
- 批准号:
24K17112 - 财政年份:2024
- 资助金额:
$ 2.29万 - 项目类别:
Grant-in-Aid for Early-Career Scientists
Molecular recognition and enantioselective reaction of amino acids
氨基酸的分子识别和对映选择性反应
- 批准号:
23K04668 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Basic research toward therapeutic strategies for stress-induced chronic pain with non-natural amino acids
非天然氨基酸治疗应激性慢性疼痛策略的基础研究
- 批准号:
23K06918 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Molecular mechanisms how arrestins that modulate localization of glucose transporters are phosphorylated in response to amino acids
调节葡萄糖转运蛋白定位的抑制蛋白如何响应氨基酸而被磷酸化的分子机制
- 批准号:
23K05758 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Design and Synthesis of Fluorescent Amino Acids: Novel Tools for Biological Imaging
荧光氨基酸的设计与合成:生物成像的新工具
- 批准号:
2888395 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Studentship
Collaborative Research: RUI: Elucidating Design Rules for non-NRPS Incorporation of Amino Acids on Polyketide Scaffolds
合作研究:RUI:阐明聚酮化合物支架上非 NRPS 氨基酸掺入的设计规则
- 批准号:
2300890 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Continuing Grant
Structurally engineered N-acyl amino acids for the treatment of NASH
用于治疗 NASH 的结构工程 N-酰基氨基酸
- 批准号:
10761044 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Lifestyle, branched-chain amino acids, and cardiovascular risk factors: a randomized trial
生活方式、支链氨基酸和心血管危险因素:一项随机试验
- 批准号:
10728925 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别:
Single-molecule protein sequencing by barcoding of N-terminal amino acids
通过 N 端氨基酸条形码进行单分子蛋白质测序
- 批准号:
10757309 - 财政年份:2023
- 资助金额:
$ 2.29万 - 项目类别: