The roles of telomerase and non-coding RNA in cancer

项目摘要 博士前阶段的这一建议将集中在生物化学和生物物理研究的酶 端粒酶端粒酶是一种核糖核蛋白，在快速分裂的细胞中维持端粒长度，从而 抵消了每一轮细胞分裂所固有的染色体缩短。没有端粒酶 当端粒长度变得非常短时，细胞经历衰老。端粒酶在大约10分钟内上调， 90%的癌症由于快速分裂的细胞需要端粒维持，使其成为一种有吸引力的药物 目标然而，直接靶向活性端粒酶的癌症治疗仍然难以捉摸。结构 人类端粒酶的功能仍然知之甚少，任何新的见解都将大大有利于人类的研究。 开发新的癌症治疗方法。本论文的研究重点是利用单分子技术 研究人端粒酶的构象和动力学。最小的人类端粒酶是由 两个亚基，一个包含四个进化保守结构域的蛋白质组分（TERT）和一个RNA 一种包含完整的非编码RNA模板的组分（TER），端粒从该模板反转 转录。端粒酶的独特之处在于它能够在一个单一的细胞周期中逆转录多个端粒重复序列。 DNA结合事件，但端粒酶如何维持这一过程的行动仍然知之甚少。 此外，关于TERT和TER在催化过程中如何相互作用，以及单个TERT结构域如何相互作用的细节， 调节端粒酶动力学，仍然未知。具体而言，关于功能和动力学的知识， 两个TERT结构域，端粒酶必需N-末端（TEN）结构域和C-末端延伸（CTE） 尤其是知之甚少。该项目将测试TEN和CTE域驱动 端粒酶通过端粒/模板杂合体的协调动态重排进行性作用， 它在每个端粒重复序列添加事件结束时的初始结合状态。单分子福斯特共振 能量转移（smFRET），与一种新的蛋白质标记方案相结合，将被用来探测蛋白质。 这些结构域在不同的功能状态下的构象和动力学。此外，计算 与斯坦福大学的Das实验室合作进行建模，将能够构建一个精细的工作模型， 人端粒酶催化过程中。这项提案的完成将有助于端粒酶的发展 靶向癌症治疗。此外，这项建议亦有助研究可再生能源计划的工作， 结构和功能在博士前阶段，以研究长非编码RNA（lncRNA）的结构和 它在博士后阶段癌症发展中的作用。学到的技术和方法 在本论文的研究将完全适合于RNA结构的研究，迄今为止， 仍然是一个难以研究的领域。拟议的博士后工作的时间集中在lncRNA结构上 考虑到lncRNA生物学的当前阶段，癌症将是理想的，总的来说，这个提议非常适合 为RNA结构如何促进癌症发展提供有价值的见解。 !

Project Summary The predoctoral phase of this proposal will focus on the biochemical and biophysical studies of the enzyme telomerase. Telomerase is a ribonucleoprotein that maintains telomere lengths in rapidly dividing cells, thereby counteracting the chromosome shortening that is inherent to each round of cell division. Without telomerase, cells undergo senescence when telomere lengths become critically short. Telomerase is upregulated in about 90% of cancers due to the need for telomere maintenance in rapidly dividing cells making it an attractive drug target. Cancer therapeutics that target active telomerase directly have remained elusive however. The structure and function of human telomerase is still poorly understood, and any new insight would greatly benefit the development of novel cancer therapeutics. The dissertation project focuses on using single molecule techniques to study the conformation and dynamics of human telomerase. The minimal human telomerase is composed of two subunits, a protein component (TERT) that contains four evolutionarily conserved domains and an RNA component (TER) which contains the integral non-coding RNA template from which telomeres are reverse transcribed. Telomerase is unique in its ability to reverse transcribe multiple telomere repeats during a single DNA binding event, but the details of how telomerase maintain this processive action remains poorly understood. Additionally, details regarding how TERT and TER interact during catalysis, and how individual TERT domains regulate telomerase dynamics, remain unknown. Specifically, knowledge about the function and dynamics of two TERT domains, the Telomerase Essential N-terminal (TEN) domain and the C-terminal extension (CTE) remain especially poorly understood. This project will test the hypothesis that the TEN and CTE domains drive the processive action of telomerase via coordinated dynamic rearrangement of the telomere/template hybrid into its initial bound state at the end of each telomere repeat addition event. Single molecule Förster Resonance Energy Transfer (smFRET), in combination with a novel protein labeling scheme, will be used to probe the conformation and dynamics of these domains during different functional states. In addition, computational modeling in collaboration with the Das lab at Stanford will enable the construction of a refined working model of human telomerase during catalysis. Completion of this proposal will aid in the development of telomerase targeted cancer therapeutics. In addition, this proposal will facilitate the smooth transition from studying RNP structure and function during the predoctoral phase, to the study of long non-coding RNA (lncRNA) structure and its roles in the development of cancer during the postdoctoral phase. The techniques and approaches learned during the dissertation research will lend themselves perfectly to the study of RNA structure, which to date has remained a difficult area to study. The timing of the proposed postdoctoral work focused on lncRNA structure and cancer will be ideal considering the current stage of lncRNA biology and overall, this proposal is well suited to provide valuable insight into how RNA structure contributes to cancer development. !