Integration Mechanism of Site Specific Non-LTR Retrotransposons

位点特异性非 LTR 反转录转座子的整合机制

• 批准号: 0950983
• 负责人: Shawn Christensen
• 金额: $ 67.8万
• 依托单位: University of Texas at Arlington
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0950983&HistoricalAwards=false
• 关键词: Integration; Mechanism; Site; Specific; Non

Retrotransposons are ubiquitous genomic parasites found in nearly all eukaryotes and constitute a major component of these genomes. Replication of retrotransposons results in chromosomal aberrations including insertions, deletions, and double stranded breaks. Upon integration into the genome, retrotransposons can affect cellular gene expression and can act as a source of new genes. Non-long terminal repeat retrotransposons (NLRs) in particular have played a central role in this process. The integration machinery of these elements is responsible for most of the reverse transcribed material in animal genomes, including short interspersed nucleotide elements (SINEs), pseudogenes, and retrogenes. For example, over 34% of the human genome is the direct result of NLR activity. In addition, NLR elements' or their progenitors' played a role in the origin of retroviruses. A detailed knowledge of the replication mechanism of NLRs is essential to understanding how retrotransposons interact with the genome. Certain aspects of element integration, especially target primed reverse transcription (a process whereby an RNA transcript is copied into a DNA strand at the site of insertion), are reasonably well understood; however, how the integration process is completed (e.g., how double stranded DNA is made from the products of target primed reverse transcription), and how NLRs can change their integration site specificity is a mystery. A mechanistic understanding of full integration has eluded study until now because of the complexity of the reaction and the lack of the right tools to address the question. In vivo systems using mutant NLR elements to the search for structure function relationships are often hard to interpret when the mutant fails to integrate. In vitro models have been missing a key RNA component that is required for complete integration to occur. This research will explore the second half of the integration reaction by restoring the missing RNA component - enabling full integration events to occur in vitro as well as in vivo. How site specific NLRs interact with DNA and change site specificity over time will also be addressed with the hopes of developing NLRs into a site specific gene targeting vector. Both aims will provide information on not only the integration mechanism of the elements used in this study but also on the integration of all NLRs. The core mechanism of integration is believed to be conserved for this class of retroelements and shares mechanistic similarities to (and evolutionary connections with) telomere elongation, group II homing introns, and retroviruses.Broader Impacts. The biochemical study of (NLRs) will be of interest to people in transposon biology, genome biology, evolutionary biology, and to those in the retroviral and biomedical fields. The University of Texas Arlington has high numbers of underrepresented groups in its graduate and undergraduate programs. This diversity is reflected in the makeup of the investigator's lab; the majority of the graduate and undergraduate researchers in the investigator's lab are from underrepresented groups. This research will be disseminated through peer reviewed journals and scientific meetings. The investigator is a guest lecturer in continuing education classes for local K-12 teachers where he talks about transposons, gene formation, and genomics; topics central to this research. These topics are also covered in the undergraduate and graduate courses the investigator teaches using research from the investigators lab.

反转录转座子是几乎所有真核生物中普遍存在的基因组寄生物，并构成这些基因组的主要组成部分。反转录转座子的复制导致染色体畸变，包括插入、缺失和双链断裂。一旦整合到基因组中，逆转录转座子可以影响细胞基因表达，并可以作为新基因的来源。特别是非长末端重复反转录转座子（NLR）在这一过程中发挥了核心作用。这些元件的整合机制负责动物基因组中的大多数逆转录物质，包括短散布核苷酸元件（西内斯）、假基因和逆转录基因。例如，超过34%的人类基因组是NLR活性的直接结果。此外，NLR元件或其祖细胞在逆转录病毒的起源中起作用。详细了解NLR的复制机制对于理解反转录转座子如何与基因组相互作用至关重要。元件整合的某些方面，特别是靶向引发的逆转录（RNA转录物在插入位点复制到DNA链中的过程），是相当好理解的;然而，整合过程是如何完成的（例如，双链DNA是如何从靶启动逆转录的产物中产生的），以及NLR如何改变它们的整合位点特异性是一个谜。由于反应的复杂性和缺乏正确的工具来解决这个问题，迄今为止，对完全整合的机械理解一直没有得到研究。当突变体不能整合时，使用突变体NLR元件来搜索结构功能关系的体内系统通常难以解释。体外模型一直缺少一个关键的RNA组分，这是完全整合发生所必需的。这项研究将通过恢复缺失的RNA组分来探索整合反应的后半部分-使完全整合事件能够在体外和体内发生。位点特异性NLR如何与DNA相互作用并随时间改变位点特异性也将被解决，希望将NLR开发成位点特异性基因靶向载体。这两个目标不仅将提供关于本研究所用要素的整合机制的信息，而且还将提供关于所有NLR整合的信息。整合的核心机制被认为是保守的这类逆转录因子和股票的机制相似性（和进化的联系）端粒延长，II组归巢内含子，和逆转录病毒。转座子生物学、基因组生物学、进化生物学以及逆转录病毒和生物医学领域的人们将对NLR的生物化学研究感兴趣。德克萨斯大学阿灵顿的研究生和本科生课程中有大量代表性不足的群体。这种多样性反映在调查员实验室的组成中;调查员实验室中的大多数研究生和本科生研究人员来自代表性不足的群体。这项研究将通过同行评审的期刊和科学会议进行传播。研究人员是当地K-12教师继续教育课程的客座讲师，他在那里谈论转座子，基因形成和基因组学;本研究的核心主题。这些主题也涵盖在本科和研究生课程的研究员教使用研究员实验室的研究。