Biogenesis and Regulation of Human Telomerase

描述（由申请方提供）：端粒酶通过添加串联端粒重复序列延长染色体末端。需要这种新的DNA合成来平衡常规DNA聚合酶在染色体末端的不完全复制中固有的DNA损失。单细胞真核生物组成型激活端粒酶并维持端粒长度的稳态。令人惊讶的是，人类体细胞没有：它们显示端粒重复序列随着增殖而逐渐缩短。胚胎、生殖系、上皮组织和造血系统中的一些人类细胞在细胞裂解物中具有可检测水平的端粒酶催化活性，但这种激活水平不足以防止所有人类组织中端粒长度随年龄的总体损失。累积的丢失最终会产生一个太短而不能保护染色体末端的重复序列，导致细胞周期的被迫退出。癌细胞显著上调端粒酶以允许无限期生长。由于这个原因，端粒酶抑制剂作为广泛有效的抗癌治疗剂具有很大的前景。端粒酶激活剂可能具有同样重要的应用，用于扩展具有由遗传、疾病、年龄或环境引起的严重短端粒的正常体细胞的更新能力。端粒酶RNA亚基（TER）作为前体表达，其必须被加工、折叠和组装成稳定的核糖核蛋白（RNP）复合物，以便在体内积累到可检测的水平。然后，该RNP招募端粒酶逆转录酶（TERT）以产生活性酶。柯林斯实验室在以前的资助期间的努力贡献了关于人类TER前体加工和RNP组装的内源性途径的开创性见解，并发现了成熟端粒酶RNP积累的缺陷，这些缺陷是X连锁和常染色体显性形式的骨髓衰竭综合征先天性角化不良的基础。下一个资助期的具体目标是解决有关体内人类端粒酶RNP积累和催化活化的知识方面的剩余空白。目的1利用人类细胞中瞬时和稳定TER表达的方法来发现和表征TER成熟和生物稳定性所需的额外RNA基序和蛋白质。目的2应用柯林斯实验室在RNA-蛋白质相互作用分析和亲和纯化方面的专业知识，以确定遗传性人类端粒酶缺陷疾病的生化缺陷。目的3研究端粒酶RNP与端粒酶逆转录酶的组装及活性。体内重建方法将与体外和体内活性测定相结合，以确定催化循环中的TER基序功能。将建立活性RNP内RNA和蛋白质结构域相互作用的生理特异性。这些研究的长期目标是了解正常细胞和疾病中端粒酶RNP的组装、催化活化和细胞调节，并利用这种理解来改善人类健康。公共卫生相关性：了解端粒酶在人体细胞和癌细胞中的生物发生和催化活化的生化特异性，将为临床操纵端粒酶以减少癌症生长或增强组织更新提供机会。此外，可以为患有由端粒酶缺乏引起的骨髓衰竭综合征的患者设计治疗方法。

DESCRIPTION (provided by applicant): Telomerase elongates chromosome ends by addition of tandem telomeric repeats. This new DNA synthesis is required to balance the loss of DNA that is inherent in the incomplete replication of chromosome ends by conventional DNA polymerases. Single-celled eukaryotes constitutively activate telomerase and maintain a homeostasis of telomere length. Surprisingly, human somatic cells do not: they show progressive shortening of the telomeric repeat array with proliferation. Some human cells in the embryo, germline, epithelial tissues, and hematopoietic system have detectable levels of telomerase catalytic activity in cell lysates, but this level of activation is insufficient to prevent an overall loss of telomere length in all human tissues with age. Cumulative loss eventually produces a repeat array that is too short to protect the chromosome end, resulting in a forced exit from the cell cycle. Cancer cells dramatically up-regulate telomerase to permit indefinite growth. For this reason, telomerase inhibitors have great promise as broadly effective anti-cancer therapeutics. Telomerase activators may have equally significant application for expanding the renewal capacity of normal somatic cells with critically short telomeres arising from genetics, disease, age, or environment. The telomerase RNA subunit (TER) is expressed as a precursor that must be processed, folded, and assembled as a stable ribonucleoprotein (RNP) complex in order to accumulate to detectable levels in vivo. This RNP then recruits telomerase reverse transcriptase (TERT) to generate the active enzyme. Collins lab efforts in previous funding periods have contributed pioneering insights about the endogenous pathway of human TER precursor processing and RNP assembly and discovered defects in the accumulation of mature telomerase RNP that underlie X-linked and autosomal dominant forms of the bone marrow failure syndrome dyskeratosis congenita. The Specific Aims of the next funding period address remaining gaps in knowledge about human telomerase RNP accumulation and catalytic activation in vivo. Aim 1 exploits methods of transient and stable TER expression in human cells to discover and characterize additional RNA motifs and proteins required for TER maturation and biological stability. Aim 2 applies Collins lab expertise in RNA-protein interaction assays and affinity purification to define the biochemical defects that underlie inherited human diseases of telomerase deficiency. Aim 3 investigates the assembly and activity of telomerase RNP with TERT. In vivo reconstitution methods will be combined with in vitro and in vivo activity assays to define TER motif functions in the catalytic cycle. The physiological specificity of RNA and protein domain interactions within the active RNP will be established. The long-term goal of these studies is to understand telomerase RNP assembly, catalytic activation, and cellular regulation in normal cells and disease and to exploit this understanding for improvement of human health. PUBLIC HEALTH RELEVANCE: Understanding the biochemical specificity of telomerase biogenesis and catalytic activation in human somatic cells and cancer cells will generate opportunities for clinical manipulation of telomerase to reduce cancer growth or enhance tissue renewal. In addition, therapies can be designed for patients with bone marrow failure syndromes arising from telomerase deficiency.