Biogenesis and Regulation of Human Telomerase

描述（由申请人提供）：端粒酶通过添加串联端粒重复序列延长染色体末端。这种新的DNA合成需要平衡传统DNA聚合酶在染色体末端不完全复制中固有的DNA损失。单细胞真核生物组成性地激活端粒酶并维持端粒长度的稳态。令人惊讶的是，人类体细胞却不是这样：它们随着增殖表现出端粒重复序列的逐渐缩短。胚胎、种系、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着年龄的增长而整体丧失。累积损失最终产生的重复序列太短，无法保护染色体末端，导致细胞周期被迫退出。癌细胞显著上调端粒酶以允许无限生长。由于这个原因，端粒酶抑制剂作为广泛有效的抗癌药物具有很大的前景。端粒酶激活剂在扩大因遗传、疾病、年龄或环境导致的端粒极短的正常体细胞的更新能力方面也有同样重要的应用。端粒酶RNA亚基（TER）作为一种前体表达，必须经过加工、折叠和组装成稳定的核糖核蛋白（RNP）复合物，才能在体内积累到可检测的水平。这个RNP然后招募端粒酶逆转录酶（TERT）来产生活性酶。在之前的资助期内，Collins实验室的努力对人类TER前体加工和RNP组装的内源性途径做出了突破性的见解，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骨髓衰竭综合征先天性角化异常的基础。下一个资助期的具体目标是解决人类端粒酶RNP积累和体内催化激活方面的知识空白。目的1利用人类细胞中瞬时和稳定的TER表达方法，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 -蛋白相互作用测定和亲和纯化方面的专业知识来确定遗传人类端粒酶缺乏疾病的生化缺陷。目的3研究端粒酶RNP与TERT的组装和活性。体内重组方法将与体外和体内活性分析相结合，以确定TER基序在催化循环中的功能。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的端粒酶RNP组装、催化激活和细胞调节，并利用这些理解来改善人类健康。公共卫生相关性：了解人类体细胞和癌细胞端粒酶生物发生和催化活化的生化特异性，将为临床操作端粒酶减少癌症生长或增强组织更新提供机会。此外，可以为端粒酶缺乏引起的骨髓衰竭综合征患者设计治疗方法。

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.