Structure, Function and Evolution of the TelomeraseRNA Subunit in Plants

本项目的总体目标是表征端粒酶RNA （TER）亚基在拟南芥中的结构、功能和相互作用。端粒酶是一种核糖核蛋白逆转录酶，它以TER为模板指导端粒DNA的合成到染色体末端。初步数据出人意料地显示，拟南芥与所有其他模式生物不同，具有两个TER亚基，TER1G7和TER5G2。这些rna表现出不同的表达水平、体外生化特性和蛋白质结合伙伴。在所有真核生物中，端粒束的维持对于维持基因组完整性和促进细胞增殖至关重要，阐明这两种拟南芥蛋白的功能和相互作用可能为端粒酶的调节和进化提供新的见解。在目的1中，我们提出了遗传和转基因的方法来研究TER1G7和TER5G2在体内的功能。我们将研究这两种rna在端粒维持中的相对贡献，以及改变这两种rna的比例所产生的影响。目的2通过体外重组活性RNP颗粒，研究TER1G7和TER5G2与TERT的相互作用。这些研究将探讨RNP的二聚化状态及其与酶活性的关系。研究人员还提出了体内实验来研究端粒酶活性水平不同的拟南芥器官中RNP的组成和寡聚现象。这项工作将解决端粒酶在植物中的功能和调控的基本问题，也可能揭示端粒酶的关键成分的复制和多样化的生理优势。该项目的学术价值在于，它将为端粒酶全酶的核心成分TER的结构、功能和进化提供新的见解。这项工作的新颖之处在于，它将拟南芥的遗传易变性与体外生物化学结合起来，探索两个TER亚基的功能和动态相互作用。这些结果不仅可能揭示单细胞生物和后生动物中端粒酶RNP生物发生和组成差异的潜在机制，而且可能揭示端粒酶调节的新模式。更广泛的影响这项工作的多学科性质为参与者提供了最前沿的RNA和蛋白质生物化学，植物生物学和遗传学方面的专业知识。这些研究将采用新的工具来研究端粒酶RNA基因的复制和多样化。特别是，由于分子进化方法尚未应用于任何模型系统的端粒研究，因此这项工作对该领域的潜在影响是很高的。16年来，培养了一大批博士后、研究生和本科生。她为高中学生和教师、本科生和研究生组织做过许多讲座，包括2003年得克萨斯农工大学科学与工程领域的女性埃塞尔·阿什沃斯·tsutsui纪念讲座。首席研究员对实验室管理有着长期的兴趣，并将于2009年参加在德克萨斯A&amp；M Mays商学院举办的首届“实验室领导最佳实践”研讨会。2002年，她是Burroughs Wellcome基金/Howard Hughes医学研究所实验室管理课程的首席研究员，2005年，她在德克萨斯州奥斯汀举行的自由基生物学和医学协会年会上为科学组织的女性发表了题为“领导实验室”的演讲。

Project Summary The overall goal of this project is to characterize the structure, function and interactions of the telomerase RNA (TER) subunit in Arabidopsis. Telomerase is a ribonucleoprotein reverse transcriptase that uses TER as a template to direct the synthesis of telomeric DNA onto chromosome ends. Preliminary data unexpectedly revealed that Arabidopsis, unlike all other model organisms, has two TER subunits, TER1G7 and TER5G2. The RNAs display different levels of expression, in vitro biochemical properties and protein binding partners. Because maintenance of telomere tracts is essential to sustain genome integrity and to promote cell proliferation in all eukaryotes, elucidating the functions and interactions of the two Arabidopsis TERs is likely to provide new insight into telomerase regulation and evolution. For Objective 1, genetic and transgenic approaches are proposed to examine the functions of TER1G7 and TER5G2 in vivo. The relative contributions of the two RNAs in telomere maintenance will be studied as well as the effect of altering the ratio of the two RNAs. Objective 2 is to study TER1G7 and TER5G2 interactions with TERT by reconstituting active RNP particles in vitro. These studies will investigate the dimerization state of the RNP and its relationship to enzyme activity. In vivo experiments are also proposed to study RNP composition and oligomerization in Arabidopsis organs with different levels of telomerase activity. This work will address fundamental aspects of telomerase function and regulation in plants, and may also shed light on the physiological advantages of duplicating and diversifying a key component of the telomerase enzyme.Intellectual Merit of the Proposed StudyThe intellectual merit of this project is that it will provide new insight into the structure, function and evolution of TER, a core component of the telomerase holoenzyme. The novel aspect of this work is that it couples the genetic tractability of Arabidopsis with in vitro biochemistry to explore the function and dynamic interactions of two TER subunits. The results may not only reveal underlying mechanisms contributing to the dramatic differences in telomerase RNP biogenesis and composition observed in single-celled organisms and in metazoa, but also may uncover new modes of telomerase enzyme regulation.Broader ImpactsThe multi-disciplinary nature of this work provides participants with expertise in cutting edge RNA and protein biochemistry, plant biology and genetics. The studies to be performed employ new tools to investigate telomerase RNA gene duplication and diversification. In particular, since molecular evolution approaches have not been applied to the study of telomeres in any model system, the potential impact of this work on the field is high. Over the past 16 years, the principal investigator has trained a large number of postdoctoral fellows, graduate and undergraduate students. She has given numerous lectures for high school students and teachers, and undergraduate and graduate student organizations, including the 2003 Texas A&M University Women in Science and Engineering Ethel Ashworth-Tsutsui Memorial Lecture. The principal investigator has a long-standing interest in laboratory management and in 2009 will be an integral participant in an inaugural "best practices in laboratory leadership" workshop to take place in the Texas A&M Mays Business School. In 2002 the principal investigator was an Instructor for the Burroughs Wellcome Fund/Howard Hughes Medical Institute Laboratory Management Course, and in 2005 she presented a lecture entitled "Leading a Laboratory" for the Women in Science organization at the Annual Meeting for the Society for Free Radical Biology and Medicine in Austin, Texas.