Negative Regulation of Telomerase in Arabidopsis

智力上的功绩。端粒是染色体的物理末端，由一种名为端粒酶的特殊核糖核蛋白(RNP)酶合成和维持。端粒酶活性是完全复制染色体末端并将其与受损的DNA区分开来所必需的。端粒酶的长期缺失会导致端到端的染色体融合。因此，端粒酶对于维持基因组的完整性是必不可少的。另一方面，如果端粒形成不当导致基因丢失，端粒酶在DNA内部双链断裂上的不适当作用可能是灾难性的。因此，端粒酶活性必须受到高度调控，并限制在自然染色体末端的末端。许多研究都集中在了解端粒酶是如何在端粒上被招募和激活的，但对其负调控机制知之甚少。这项研究的总体目标是检验在模式植物拟南芥中抑制端粒酶功能的机制。Shippen实验室的初步研究揭示了拟南芥端粒酶复杂而新颖的调控途径，该途径涉及由不同蛋白质亚基和不同端粒酶RNA(TER)亚基组成的多个端粒酶。TER1 RNP是端粒酶的正向调节因子，与酵母和脊椎动物中的TER1 RNP一样，它需要在体内维持端粒。相反，TER2 RNP是端粒酶活性和端粒维持的新的负调控因子。TER2被拼接形成功能未知的TER2s。目标1将研究TER2的生物发生和功能。目标2将研究TER2 RNP在细胞周期中的动态，以了解为什么该RNP不能有效地与染色体末端接合。目标3将开发一种高效的新的端粒形成实验来研究端粒酶如何在染色体内部断裂时发挥作用。这项研究的结果将加深对端粒酶与染色体相互作用如何调节以促进基因组稳定的理解。除了对促进染色体完整性的基本机制有新的了解外，这项工作的更广泛的影响是，它将为一名博士后研究员和两名植物分子生物学和生物化学研究生提供研究机会，使他们能够在基因组研究的前沿工作。目前的NSF研究还将为德克萨斯农工大学和休斯顿大学市中心分校的本科生提供研究机会。在她的职业生涯中，Shippen博士为高中生和教师以及本科生和研究生组织做过多次演讲，包括德克萨斯农工大学理工科女性Ethel Ashworth-Tsutsui纪念讲座。希彭博士对实验室管理有着长期的兴趣。2002年，她担任Burroughs Wellcome基金/Howard Hughes医学院实验室管理课程的讲师。2008-2009年，希彭博士在哈佛大学休假，在那里她参与了一个由NSF资助的项目，该项目旨在定义实验室管理的最佳实践。这项研究正在进行中，并已导致开发了一门关于建立科学关系的新研究生课程，以及一项针对博士后和年轻PI的指导计划，为指导研究团队面临的常见问题提供实际解决方案。Shippen实验室的所有成员都从这项实验室管理研究中受益，以发展领导技能并分担同事的科学发展责任。

Intellectual Merit. Telomeres are the physical ends of chromosomes and they are synthesized and maintained by a specialized ribonucleoprotein (RNP) enzyme called telomerase. Telomerase activity is required to fully replicate the chromosome terminus and to distinguish it from damaged DNA. The prolonged absence of telomerase leads to end-to-end chromosome fusions. Thus, telomerase is essential to sustain genome integrity. On the other hand, inappropriate action of telomerase at internal double-strand breaks in the DNA can be catastrophic if telomeres form inappropriately to cause gene loss. Consequently, telomerase activity must be highly regulated and confined to the tips of natural chromosome ends. Much research has focused on understanding how telomerase is recruited and activated at telomeres, but little is known about mechanisms of negative regulation. The overall goal of this research is to examine mechanisms that suppress telomerase function in the model plant Arabidopsis. Preliminary studies from the Shippen lab reveal a complex and novel regulatory pathway for Arabidopsis telomerase that involves multiple telomerase enzymes composed of different protein subunits and different telomerase RNA (TER) subunits. The TER1 RNP is a positive regulator of telomerase, and like its counterparts in yeast and vertebrates, it is required to maintain telomeres in vivo. In contrast, the TER2 RNP is a novel negative regulator of telomerase activity and telomere maintenance. TER2 is spliced to form TER2s, whose function is unknown. Objective 1 will examine the biogenesis and function of TER2s. Objective 2 will study the dynamics of the TER2 RNP during the cell cycle to understand why this RNP cannot productively engage the chromosome terminus. Objective 3 will exploit a highly efficient new telomere formation assay to study how telomerase action is restricted at internal chromosome breaks. The results of this research will deepen understanding of how telomerase interaction with chromosomes is regulated to promote genome stability.Broader Impacts. In addition to shedding new light on fundamental mechanisms that promote chromosome integrity, the broader impact of this work is that it will provide research opportunities for a postdoctoral fellow and two graduate students in plant molecular biology and biochemistry, allowing them to work at the forefront of genome research. The current NSF study will also provide research opportunities for undergraduate students from Texas A&M and the University of Houston-Downtown, a URM campus. Throughout her career, Dr. Shippen has given numerous lectures for high school students and teachers, and undergraduate and graduate student organizations, including the Texas A&M University Women in Science and Engineering Ethel Ashworth-Tsutsui Memorial Lecture. Dr. Shippen has a long-standing interest in laboratory management. In 2002 she was an Instructor for the Burroughs Wellcome Fund/Howard Hughes Medical Institute Laboratory Management Course. In 2008-2009, Dr. Shippen was on sabbatical leave at Harvard University, where she worked on an NSF-funded project to define best practices in laboratory management. This study is ongoing and has already led to the development of a new graduate course on building scientific relationships and a mentoring program for postdocs and young PIs that provides practical solutions to common problems faced in guiding a research team. All the members of the Shippen lab benefit from this lab management research to develop leadership skills and shared responsibility for the scientific development of their colleagues.