NSF Award
2427326
While the distributions of thousands of proteins within cells have been characterized over the last half century, facilitated by tools such as specific antibodies, immunofluorescent staining, and molecular fluorescent tags, much less is known about where and how RNA molecules are transported and localized. Nonetheless, proper RNA localization is essential for organism development, and aberrant localization can result in lethality. Although the distribution patterns of a handful of RNAs in various organisms have been investigated in detail, many remain uncharacterized. Moreover, for several RNAs their localization pattern has been reported but the transportation mechanism(s) remain(s) unknown. Part of this knowledge deficit is attributable to the challenging technical aspects of detecting RNAs in vivo, which requires multidisciplinary expertise to design and deliver the RNA detection probes into the cell or tissue of interest, as well as advanced live-cell imaging and microscopy. This project will develop computational and experimental tools to facilitate quick and sensitive studies of RNA transport and localization in real time. Of particular interest, vesicles are subcellular structures that have been well described regarding their roles in intra- and inter-cellular transport of proteins but only recently have been linked also to intracellular RNA trafficking. These tools will be applied to co-visualize RNAs and transport vesicle markers and determine the role vesicles play in long-range RNA transport in a favorite cell biology model system, the fruit fly egg. The Broader Impacts of the work include its intrinsic merit, as all cells need to transport and localize RNAs, and the development of tools that will be of wide utility to the community. Additional impact will come from the training of undergraduate students in research at an undergraduate institution that has limited research opportunities.<br/><br/>Early studies of RNA localization in the fruit fly embryo revealed that proper mRNA localization is essential for correct tissue differentiation. Numerous trafficking mechanisms have since been elucidated, with many operating in Drosophila melanogaster germ cells and conserved in other cell types, including neurons. Vesicular trafficking factors have been implicated in long-range RNA transport in neurons (Rab5, Rab7, and Rab11), but to date, it is unknown how widespread this novel mechanism is. The proposed studies will identify connections between maternally deposited RNAs and intracellular vesicular trafficking via Rab family members in the fruit fly egg chamber. To achieve this, a Python-based software package will be developed for easy and accessible design of custom probes by integrating the PI’s previously developed probe design scripts and including flexible probe options for any RNA target from any model organism. Probe candidates for a given target will be ranked within the program based on a hybridization efficiency factor calculated using a math model that accounts for target structure, probe backbone chemistry, addition of denaturing reagents, and temperature. Finally, with the new RNA probes in hand, the effects of modulating the expression and/or function of nine Rab proteins on the transport and localization of eleven maternal mRNAs will be investigated throughout fruit fly oogenesis. These ex vivo studies will provide proof of principle for optimization and validation of the software package and will shed light on the role of vesicular trafficking in intracellular RNA localization in real time.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
