Date of Award
Spring 5-3-2023
Degree Type
Thesis
Degree Name
Master of Science in Integrative Biology (MSIB)
Department
Biology
Committee Chair/First Advisor
Brandon Carpenter
Major Professor
Anton Bryantsev
Second Committee Member
Susan M. Smith
Third Committee Member
Carol Chrestensen
Abstract
The structural organization of the cell nucleus poses many intriguing questions. One of them is the organization of nuclear domains (ND), sharp-bordered nuclear compartments concentrating a range of nuclear proteins. In this study, we used the recently discovered B-body as a model to investigate the mechanisms governing the formation of NDs. The nuclear B-body forms in the cellular precursors of flight muscles in developing Drosophila pupae. Prior to myoblast fusion, the splicing factor Bruno (Bru) concentrates into a single, large B-body but transitions into multiple smaller speckles in a diffused nuclear pattern after the commencement of myogenesis. We hypothesized that the B-body must contain an RNA scaffold because Bru association with B-bodies is RNase-sensitive. Using Immuno-FISH, we identified a lncRNA co-localizing with the B-body; this RNA is the heat shock RNA omega (hsrω). Next, we tested the requirement of protein and RNA components for the integrity of the B-body. Genetic knockdown of the protein Bru did not perturb the size of B-body, as revealed by hsrω FISH. In contrast, when hsrω was removed via chromosomal deletions, the structural stability of the B-body was severely affected. Since hsrω is expressed in a broader range of tissues than Bru, we conducted a misexpression study to test if it is possible to reconstitute the B-body outside flight muscles. Nuclei of the midgut epithelium are similar to the polyploid nuclei to flight muscle progenitors that express hsrω; however, ectopically expressed Bru did not accumulate to form a B-body in them. Lastly, we found that flies with reduced hsrω showed normal IFM development, flight ability, and viability similar to their wild-type counterparts. In summary, our study demonstrates the importance of an RNA scaffolding for the B-body and highlights the importance of an additional mechanism to enable protein trafficking and accumulation at B-bodies. Meanwhile, the functional significance of B-bodies remains elusive and will require additional studies.