Identification of endogenous TERRA-ribonucleoprotein components and characterization of their function in telomere biology

端粒形成真核染色体的末端。一组端粒特异性成分作为基因组完整性的守护者和肿瘤发生的抑制者发挥着基本的作用。最近，一种新的和意想不到的端粒成分被描述：含有RNA的端粒重复序列(Terra)。一些观察结果表明，Terra在维持端粒完整性方面发挥了重要作用。然而，关于这种长的非编码RNA的精确调控知识仍然很少。为了解决这个问题，这个建议旨在描述Terra结合蛋白在Terra和端粒新陈代谢中的功能。因此，内源性Terra及其相关因子，也称为Terra-核糖核蛋白复合体(Terra-RNPs)，将从人类细胞系中分离出来。接下来，将通过质谱学鉴定Terra结合蛋白。通过对识别的因子进行遗传操作，将评估它们在Terra新陈代谢和端粒维持中的作用。这将阐明Terra是如何影响染色体稳定性的，并将进一步确定与端粒完整性和抑制癌变有关的新因素。这不仅可能揭示非编码RNA如何控制端粒/基因组完整性的新机制，而且还可以作为其他非编码转录本的作用模式和调节的范例。此外，对调节Terra并进而控制端粒维持的因素的表征可能会确定癌症治疗的新的和有希望的靶点。最后，表征Terra-RNPs将扩大我们对与端粒功能障碍相关的人类疾病的分子机制的了解。

Telomeres form the ends of eukaryotic chromosomes. A set of telomere-specific components plays fundamental roles as guardians of genome integrity and suppressors of tumorigenesis. Recently, a novel and unexpected telomere component has been described: the telomeric repeat containing RNA (TERRA). Several observations point towards an essential role of TERRA in the maintenance of telomere integrity. Nevertheless, precise knowledge about the regulation of this long non-coding RNA is still sparse. To address this issue, this proposal aims at the characterization of TERRA-binding proteins with regard to their function in TERRA and telomere metabolism. Hereby, endogenous TERRA and its associated factors, also called TERRA-ribonucleoprotein complexes (TERRA-RNPs), will be isolated from a human cell line. Next, TERRA-binding proteins will be identified by mass spectrometry. By genetic manipulation of the identified factors, their role in TERRA-metabolism as well as telomere maintenance will be assessed. This will elucidate how TERRA impinges on chromosome stability and will further identify new factors implicated in telomere integrity and the suppression of carcinogenesis. Not only may this uncover a novel mechanism of how a non-coding RNA can control telomere/genome integrity, but also serve as a paradigm for the mode of action and regulation of other non-coding transcripts. Furthermore, the characterization of factors that modulate TERRA and in turn control telomere maintenance may identify novel and promising targets for cancer therapy. Finally, characterizing TERRA-RNPs will expand our knowledge on the molecular mechanisms causative for human diseases associated with telomere dysfunction.