Structure, Function and Evolution of the TelomeraseRNA Subunit in Plants

植物端粒酶RNA亚基的结构、功能和进化

• 批准号: 0843399
• 负责人: Dorothy Shippen
• 金额: $ 54万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843399&HistoricalAwards=false
• 关键词: Structure; Function; Evolution; TelomeraseRNA; Subunit

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.

本项目的总体目标是研究拟南芥端粒酶RNA（TER）亚基的结构、功能和相互作用。端粒酶是一种核糖核蛋白逆转录酶，其使用TER作为模板来指导端粒DNA合成到染色体末端。初步的数据出乎意料地显示，拟南芥，不像所有其他模式生物，有两个TER亚基，TER 1G 7和TER 5G 2。RNA显示出不同的表达水平、体外生物化学性质和蛋白质结合伴侣。由于端粒区的维持对于维持基因组完整性和促进所有真核生物的细胞增殖至关重要，因此阐明两个拟南芥TERs的功能和相互作用可能为端粒酶调控和进化提供新的见解。对于目标1，提出了遗传和转基因方法来检查TER 1G 7和TER 5G 2在体内的功能。将研究两种RNA在端粒维持中的相对贡献以及改变两种RNA的比率的影响。目的二是通过体外重组活性RNP颗粒研究TER 1G 7和TER 5G 2与TERT的相互作用。这些研究将调查RNP的二聚化状态及其与酶活性的关系。在体内实验也提出了研究RNP的组成和寡聚化在拟南芥器官与不同水平的端粒酶活性。这项工作将解决端粒酶的功能和调节在植物中的基本方面，也可能揭示了复制和多样化的端粒酶的关键组成部分的生理优势。拟议研究的智力价值这个项目的智力价值是，它将提供新的见解的结构，功能和进化的TER，端粒酶全酶的核心组成部分。这项工作的新颖之处在于它将拟南芥的遗传易处理性与体外生物化学相结合，以探索两个TER亚基的功能和动态相互作用。这些结果不仅可能揭示导致在单细胞生物和后生动物中观察到的端粒酶RNP生物发生和组成的巨大差异的潜在机制，而且还可能揭示端粒酶酶调节的新模式。即将进行的研究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，由于分子进化的方法还没有被应用到任何模型系统中的端粒的研究，这项工作对该领域的潜在影响是高的。在过去的16年里，主要研究者培养了大量的博士后研究员，研究生和本科生。她为高中学生和教师以及本科生和研究生组织举办了许多讲座，包括2003年德克萨斯州A M大学科学与工程女性埃塞尔·阿什维尔-Tsutsui纪念讲座。首席研究员对实验室管理有着长期的兴趣，2009年将成为德克萨斯州A M Mays商学院首届“实验室领导力最佳实践”研讨会的重要参与者。2002年，主要研究者是Burroughs Wellcome基金/霍华德休斯医学研究所实验室管理课程的讲师，2005年，她在德克萨斯州奥斯汀举行的自由基生物学和医学学会年会上为女性科学组织做了题为“领导实验室”的演讲。