Biogenesis and Regulation of Human Telomerase

端粒酶通过增加串联的端粒重复序列来延长染色体末端。这种新的DNA 合成是必需的，以平衡DNA的丢失，这是由于不完全复制所固有的 染色体末端由传统的DNA聚合酶终止。单细胞真核生物结构性激活 端粒酶和维持端粒长度的动态平衡。令人惊讶的是，人类体细胞并不： 它们显示随着增殖，端粒重复序列逐渐缩短。一些人体细胞在 胚胎、生殖系、上皮组织和造血系统有可检测到的水平 细胞裂解物中的端粒酶催化活性，但这种激活水平不足以防止 随着年龄的增长，所有人体组织的端粒总长度都会减少。累计亏损最终会产生 重复排列，太短而不能保护染色体末端，导致被迫退出细胞 周而复始。癌细胞戏剧性地上调端粒酶以允许无限生长。因为这个原因， 端粒酶抑制剂作为广泛有效的抗癌治疗药物前景广阔。端粒酶 激活器在扩展Normal的续订能力方面可能有同样重要的应用 端粒极短的体细胞，端粒极短是由遗传、疾病、年龄或环境引起的。 端粒酶RNA亚单位(TER)被表达为必须被加工、折叠、 并组装成稳定的核糖核蛋白(RNP)复合体，以便累积到可检测 体内水平。该RNP然后招募端粒酶逆转录酶(TERT)来产生活性 酵素。柯林斯实验室在之前的资助时期所做的努力为我们提供了关于 人类TER加工和RNP生物发生的内源途径以及在 X连锁和常染色体显性骨髓的端粒酶RNP生物发生 先天性角化不良衰竭综合征。 下一个供资阶段的具体目标是解决关于以下方面的知识方面的重大剩余差距 人端粒酶RNP的生物发生和催化激活。目标1利用瞬变和 在人类细胞中稳定表达TER以发现和鉴定额外的RNA基序和 指导端粒酶RNP生物发生的相互作用的蛋白质。AIM 2将柯林斯实验室的专业知识应用于RNA 和蛋白质亲和纯化以阐明人类疾病缺陷的分子机制 端粒酶RNP的生物发生。目的3研究端粒酶RNP与TERT的组装以形成 催化活性酶。体内重建方法将与催化试验相结合 体外和体内活性阐明人TER-TERT相互作用的生理特异性 TER基序在端粒重复序列合成催化循环中的作用。的长期目标是 这些研究旨在了解端粒酶的生物发生、催化激活和细胞调控。 正常细胞和疾病，并利用这一认识来改善人类健康。

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 level 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 processing and RNP biogenesis and discovered defects in telomerase RNP biogenesis 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 crucial remaining gaps in knowledge about human telomerase RNP biogenesis and catalytic activation. Aim 1 exploits methods of transient and stable TER expression in human cells to discover and characterize additional RNA motifs and interacting proteins that direct telomerase RNP biogenesis. Aim 2 applies Collins lab expertise in RNA and protein affinity purification to elucidate the molecular mechanisms of human disease defects in telomerase RNP biogenesis. Aim 3 investigates telomerase RNP assembly with TERT to form the catalytically active enzyme. In vivo reconstitution methods will be combined with assays of catalytic activity in vitro and in vivo to elucidate the physiological specificity of human TER-TERT interaction and TER motif functions in the catalytic cycle of telomeric repeat synthesis. The long-term goal of these studies is to understand telomerase biogenesis, catalytic activation, and cellular regulation in normal cells and disease and to exploit this understanding for improvement of human health.