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

• Applicant: Dr. Andreas Ettinger
• Subject Area: Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406108326

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.

• DFG Programme: Research Grants