Biogenesis and Regulation of Human Telomerase

端粒酶通过添加串联端粒重复序列来延长染色体末端。这种新的DNA 合成需要平衡DNA的损失，这是在不完全复制中固有的。 染色体末端的DNA聚合酶。单细胞真核生物组成型激活 端粒酶和维持端粒长度稳态。令人惊讶的是，人体细胞没有： 它们显示随着增殖端粒重复序列逐渐缩短。一些人体细胞 胚胎、生殖系、上皮组织和造血系统具有可检测水平的 细胞裂解物中的端粒酶催化活性，但这种活化水平不足以防止 所有人体组织中端粒长度随年龄增长而总体丧失。累积损失最终会产生 太短而不能保护染色体末端的重复序列，导致从细胞中强制退出 周期癌细胞显著上调端粒酶以允许无限期生长。基于这个理由， 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 活化剂可以具有同样重要的应用，用于扩大正常的再生能力， 由于遗传、疾病、年龄或环境而导致端粒短的体细胞。 端粒酶RNA亚基（TER）作为前体表达，其必须被加工，折叠， 并组装成稳定的核糖核蛋白（RNP）复合物，以积累到可检测的水平。 体内水平。然后，该RNP招募端粒酶逆转录酶（TERT）以产生活性的 酵素柯林斯实验室在以前的资助期内的努力，对 人TER加工和RNP生物合成的内源性途径，并发现 X连锁和常染色体显性形式骨髓端粒酶RNP生物发生 先天性角化不良 下一个供资期的具体目标解决了在以下方面的知识方面仍然存在的关键差距： 人端粒酶RNP生物合成和催化活化。目标1利用瞬态和 在人细胞中稳定TER表达以发现和表征另外的RNA基序， 指导端粒酶RNP生物合成的相互作用蛋白。目标2应用柯林斯实验室在RNA方面的专业知识 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生物发生。目的3研究端粒酶RNP与端粒酶逆转录酶的组装， 催化活性酶体内重建方法将与催化活性测定相结合。 体外和体内活性，以阐明人TER-TERT相互作用的生理特异性 和TER基序在端粒重复合成的催化循环中起作用。的长期目标 这些研究的目的是了解端粒酶的生物发生、催化活化和细胞调控， 正常细胞和疾病，并利用这种理解来改善人类健康。

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.