Biogenesis and Regulation of Human Telomerase

描述(申请人提供)：端粒酶通过增加串联端粒重复来延长染色体末端。这种新的DNA合成是为了平衡传统DNA聚合酶对染色体末端的不完全复制所固有的DNA损失。单细胞真核细胞组成激活端粒酶并维持端粒长度的动态平衡。令人惊讶的是，人类体细胞没有：它们显示出随着增殖而逐渐缩短的端粒重复序列。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到的端粒酶催化活性，但这种激活水平不足以防止所有人类组织随着年龄的增长而整体失去端粒长度。累积丢失最终会产生重复排列，该重复排列太短而不能保护染色体末端，导致被迫退出细胞周期。癌细胞戏剧性地上调端粒酶以允许无限生长。出于这个原因，端粒酶抑制剂作为广泛有效的抗癌治疗药物具有很大的前景。端粒酶激活剂在扩大因遗传、疾病、年龄或环境引起的端粒极短的正常体细胞的更新能力方面可能具有同样重要的应用。端粒酶RNA亚单位(TER)被表达为前体，必须经过加工、折叠和组装成稳定的核糖核蛋白(RNP)复合体，才能在体内积累到可检测的水平。然后，这个RNP招募端粒酶逆转录酶(TERT)来产生活性酶。柯林斯实验室在之前的资助时期所做的工作为人类TER前体处理和RNP组装的内源性途径提供了开创性的见解，并发现了成熟端粒酶RNP积累中的缺陷，这种缺陷是X连锁和常染色体显性形式的先天性骨髓角化不良综合征的基础。下一个资助期的具体目标是解决关于人体端粒酶RNP积累和体内催化激活的剩余知识空白。目的1利用在人类细胞中瞬时和稳定表达TER的方法，发现和鉴定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来确定导致人类端粒酶缺乏的遗传性疾病的生化缺陷。目的3研究端粒酶RNP在TERT中的组装和活性。体内重组方法将与体外和体内活性测定相结合，以确定TER基序在催化循环中的功能。活性RNP中RNA和蛋白质结构域相互作用的生理特异性将被建立。这些研究的长期目标是了解端粒酶RNP在正常细胞和疾病中的组装、催化激活和细胞调节，并利用这一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中端粒酶生物发生和催化激活的生化特性，将为临床操作端粒酶以减少癌症生长或促进组织更新创造机会。此外，可以为端粒酶缺乏症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.