RNA localizations of proteins that interact with the chromosome passenger complex

Thesis (M.A.)--Boston UniversityIn the last twenty years work on chromatin binding non-coding RNAs has revealed much of their biology, but how mitosis affects these nuclear ncRNA transcripts is less well understood. This study examined the behavior of several ncRNAs through mitosis. It is understood that Aurora B Kinase, or AURKB, plays a role in the dispersal of the ncRNA Xist from chromatin in mitosis, but our experiments probed the mechanism of this interaction. Using fluorescent microscopy to visualize the location of both the ncRNA and the protein required the refinement of an IF/FISH protocol. Furthermore, other ncRNAs were identified in the literature in order to examine if they are similarly governed by AURKB, and the localization of the other ncRNAs was compared throughout mitosis to that of Xist. An RNAi screen was used to identify that MCAK, a known target of AURKB’s kinase activity, is another player in the dispersal of Xist. Our results also demonstrated that nuclear envelope breakdown precedes the release of ncRNAs into the cytoplasm, and that MCAK knockdown delays the dispersal of Xist and may also impact LINE1. We were also able to visualize AURKB and Xist throughout mitosis. This study developed our understanding of daughter cell inheritance of ncRNA mediated transcriptional silencing by showing that multiple ncRNAs disperse from the nucleus after nuclear envelope breakdown in prophase, and also that MCAK is implicated in Xist dispersal. These are important steps towards a comprehensive understanding of how the chromosomal passenger complex affects ncRNA transcripts throughout mitosis, and how the complex plays a role in retention of silencing in daughter cells by governing mitotic dispersal of ncRNA. We demonstrated ncRNA dispersal is needed for successful mitotic division, and must take place to advance past metaphase