Function of Satellite RNA Transcription for Centromere Assembly and Mitotic Spindle Formation

着丝粒DNA序列通过募集将染色体附着到纺锤体微管的动粒复合体，在有丝分裂中对染色体分布起重要作用。影响着丝粒形成和有丝分裂的分子途径直接与细胞分裂的结果相关联。在减数分裂或早期发育中，染色体的异常分离是遗传疾病如三体的原因，或者可能导致发育停滞和流产。在成年生物体中，不能正确分离姐妹染色单体可导致染色体重排，并导致肿瘤转化和肿瘤形成。尽管它们具有中心功能，但定义着丝粒身份和控制着丝粒组装的分子机制仍然没有很好的定义。已经提出了RNA组分的潜在作用，但其分子机制及其背后的意义仍然未知。在这里，我建议调查着丝粒RNA（cenRNA）转录的功能，着丝粒组装和阐明cenRNA在有丝分裂纺锤体形成和着丝粒建立的潜在作用。为此，我计划应用新的活细胞共聚焦显微镜方法和分子和药理学扰动，并利用小鼠植入前胚胎和小鼠胚胎干细胞（ESCs）作为模型系统来研究定义着丝粒身份的表观遗传变化。该项目的目标是建立在培养细胞中的活细胞cenRNA成像方法的基础上，并将其扩展到小鼠植入前胚胎作为模型系统，以解决卫星RNA动力学和cenRNA加工如何调节着丝粒组装的问题。为此，我建议（1）用基于CRISPR的活细胞RNA跟踪确定细胞周期期间着丝粒核糖核蛋白组装的空间和时间动态，（2）确定cenRNA的剪接是否以及如何在分子和细胞生物学水平上调节着丝粒组装，以及（3）确定cenRNA在早期发育中用于着丝粒建立的功能，利用卵母细胞和植入前小鼠胚胎作为定义着丝粒身份的基本表观遗传重排的模型系统。在发育和疾病中，染色体的不正确分布与遗传性疾病或肿瘤形成中的肿瘤转化有关。因此，了解cenRNA如何影响染色体分离将有助于深入了解有丝分裂正常进行的机制，以及如何可能靶向或改善疾病中的错误有丝分裂。

Centromeric DNA sequences play an essential role for chromosome distribution in mitosis by recruiting the kinetochore complex that attaches chromosomes to spindle microtubules. Molecular pathways that influence centromere formation and mitosis are directly coupled to the outcome of cell division. During meiosis or in early development, aberrant segregation of chromosomes is a cause of genetic disorders like trisomies, or can lead to developmental arrest and miscarriage. In the adult organism, failure to correctly segregate sister chromatids can lead to chromosomal rearrangements and result in neoplastic transformation and tumor formation.Despite their central function, the molecular mechanisms defining centromere identity and governing centromere assembly remain not well defined. A potential role for an RNA component has been suggested, but the molecular mechanisms and their implications behind, remain unknown. Here, I propose to investigate the functions of centromeric RNA (cenRNA) transcription for centromere assembly and elucidate potential roles of cenRNA in mitotic spindle formation and centromere establishment. To this end, I plan to apply novel live-cell confocal microscopy approaches and molecular and pharmacological perturbation and utilize the mouse pre-implantation embryo and mouse embryonic stem cells (ESCs) as model systems to study the epigenetic changes defining centromere identity. The goal of this project is to build on approaches for live-cell cenRNA imaging in cultured cells and and extend those to the mouse pre-implantation embryo as a model system to address the question how satellite RNA dynamics and cenRNA processing regulate centromere assembly. To this end, I propose to (1) determine spatial and temporal dynamics of centromeric ribonucleoprotein assembly during the cell cycle with CRISPR-based live-cell RNA tracking, (2) determine if and how splicing of cenRNAs regulates centromere assembly on a molecular and cell biological level, and (3) determine the function of cenRNA for centromere establishment in early development, utilizing oocytes and the pre-implantation mouse embryo as model systems for fundamental epigenetic rearrangements defining centromere identity. In development and disease, incorrect distribution of chromosomes is linked with hereditary disorders or neoplastic transformation in the case of tumor formation. Therefore, understanding how cenRNA impacts chromosome segregation will lead to insights into the mechanisms by which mitosis progresses normally and how to possibly target, or ameliorate, faulty mitosis in disease.