The objective of this proposal is to investigate mechanisms of RNA localization in Drosophila, as a means of understanding cytoplasmic organization and its role in gametogenesis. RNAs become localized in oocytes and other cells via interactions with the cytoskeleton, although the precise molecular interactions that accomplish localization are not known. In order to advance our understanding of RNA localization, we will take two approaches. One focuses on dissecting the function of swallow (swa), a gene involved in multiple RNA localization events. There is good circumstantial evidence that swa interacts with both microtubules and actin filaments, and we will (1) Examine the involvement of swa in a variety of cytoskeletal-based events during oogenesis. This will include testing a requirement for swa in other cytoskeletal-based processes of oogenesis, and testing models for swa function to determine causes for the range of phenotypes associated with swa mutations. We will determine if some or all of the defects associated with swa mutations are directly due to failures in swa function. Alternatively, we may discover that some swa phenotypes are indirectly the result of RNA localization defects. These experiments will reveal a wealth of information about swa functioning. Our second goal is to investigate mechanisms of RNA localization on a larger scale, by identifying novel localized RNAs, and characterizing their requirements for localization. To this end we will: (2) Use a combination of bioinformatics, biochemical fractionations and microarray analyses to identify novel localized RNAs. The distribution of candidate RNAs in ovaries will be assayed by in situ hybridization and localized RNAs will be further tested for a dependence on swa and microtubules. Overall, these studies are designed to answer some basic questions regarding both swa function in particular and RNA localization in general. For example, does swa influence RNA localization via modulation of cytoskeletal function? How can the variety of defects associated with swa mutations be explained? How many RNAs are dependent on swa for their localization? Can discrete pathways of RNA localization be defined? This research is of particular interest because it is becoming clear that many characteristics of RNA localization are highly conserved, and this type of cellular organization is likely to be essential to the functioning of all cells.
This project will investigate the role of the swallow gene in RNA localization and will identify novel localized RNAs in Drosophila for future studies. RNA localization is a widespread and essential mechanism for establishing cell polarity in a variety of cells, and a failure to establish or maintain cell polarity, is associated with a number of human diseases, including cancer. Information gained by this project will contribute to our overall understanding of cell organization and function and therefore our understanding of human health.
| Pokrywka, Nancy Jo; Zhang, Huadi; Raley-Susman, Kathleen (2014) Distinct roles for hu li tai shao and swallow in cytoskeletal organization during Drosophila oogenesis. Dev Dyn 243:906-16 |
| Pokrywka, Nancy Jo (2013) Live imaging of GFP-labeled proteins in Drosophila oocytes. J Vis Exp : |
