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。
项目成果
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