Intellectual Merit: The long-term goal of this research is to understand the molecular pathways that govern mRNA-specific rates of degradation, a key step in the control of how much protein can be produced from any one mRNA. mRNA molecules only exist for limited amounts of time before they are targeted for degradation by specific cellular machinery. Because the protein synthesis machinery can repeatedly translate a single mRNA molecule to produce multiple proteins, the lifespan of an mRNA determines how much protein will be produced from that mRNA. Decay rates of individual mRNAs in eukaryotic cells can vary by more than two orders of magnitude, and modulation of mRNA decay rates is an efficient method to rapidly alter protein expression in response to environmental or intracellular changes. This regulation is often mediated by interactions with elements in the 3' untranslated regions (UTRs) of mRNAs. Several types of RNA binding proteins are known to bind these 3' UTR elements, yet little is known of the mechanisms by which the binding of such proteins leads to functional changes in the behavior of the mRNAs. The Puf protein family is a widely conserved group of eukaryotic RNA binding proteins that control critical decisions in stem cell maintenance, cell development and differentiation, neuronal plasticity and long-term memory by binding and modulating the metabolism of target mRNAs. The yeast Saccharomyces cerevisiae contains six Puf proteins, providing a good model system to study Puf protein specificity and regulatory function. This research has already: 1) identified several mRNA targets of Puf protein regulation, 2) established that Puf3p mediates rapid mRNA decay by stimulating deadenylation and decapping, 3) identified several mRNA decay factors that interact with Puf3p, and 4) provided the first experimental evidence that Puf protein activity is dependent on growth conditions. The main goal of this project is to analyze the mechanisms by which the yeast Puf proteins differentially regulate the stability of their target mRNAs in rapid response to changing environmental conditions. Based on preliminary evidence, the hypothesis is that Puf protein activity is regulated by a post-translational mechanism. The research objectives will be accomplished using a combination of RNA decay analysis, co-immunoprecipitation studies, cell microscopy, and mass spectrometry. This work will not only provide valuable insight into the condition-specific regulation of Puf proteins and the mechanisms by which Pufs influence mRNA stability, but should have a broad impact on understanding the important developmental and neural requirements for Pufs and 3' UTR control in other eukaryotes as well.
Broader Impacts: Funding for this project will not only promote intellectual advancement, but will also support the Ph.D. training and research of an African American student in the lab. Such training and support will further her development into a role model for other under-represented minorities in the molecular sciences, especially given that this research will be performed at an urban campus with a higher than average population of minority students. In addition, project funding will support the training of thesis Master's students, non-thesis Master's students, and undergraduate students. By introducing new techniques to the lab, including cell microscopy and mass spectrometry, this project will also enhance the future experimental capabilities and potential for this research program. Finally, project support will enhance opportunities to engage diverse populations of high school students from around the state in workshops and discussions to encourage them to pursue careers in the sciences.
