Gene expression is controlled at many levels, including the processing of RNAs. There remain many questions as to the mechanisms by which environmental inputs (stimuli) lead to changes in RNA processing. The research to be pursued in this project deals with one possible mechanism, involving the role of one RNA processing factor (CPSF30) in regulated gene expression in plants. This work is inspired by prior observations that mutant plants that do not make the protein have an interesting range of phenotypes, and that the protein itself is regulated by a central component of cellular sensory systems. This project will combine genetic, biochemical, molecular, and systems approaches to understand the interplay between cellular signaling and the function of CPSF30. The outcome of the research component of the project will be a better understanding of the ways in which stimuli can cause changes in the processing of RNAs in the plant cell. This will in turn contribute to a better understanding of the ways by which plants respond to environmental cues and disease-causing organisms. This project will have an impact that extends significantly beyond an understanding of gene expression in plants. The expected results will be of help in developing new genetic and biotechnological strategies for improving the performance of crop plants in adverse conditions. This project will involve postdoctoral scientists, graduate students, and undergraduate students. There is a decided cross-disciplinary nature to this project, in that it involves molecular/biochemical, whole-plant, and systems biology approaches. As such, these studies will help to prepare trainees to make contributions in a scientific field (plant biology) that is becoming more cross-disciplinary. Participants (including PIs) will participate in community programs so as to promote interest in and appreciation of science in the general public.
In order for plants to respond to different environmental stimuli or developmental cues, they must be able to change their gene expression program so as to best adapt to changing conditions. Gene expression can be changed in many ways; one such alteration involves the ways in which messenger RNAs are processed. This project addresses important questions concerning one such mode of regulation, termed alternative polyadenylation, or APA. While APA is known to be important, little is known about how APA is connected with stimuli – in other words, how developmental cues or different stresses trigger APA. The goals of this project were to test two hypotheses that would provide insight into such connections. The first hypothesis held that a previously-identified interaction between a subunit (termed CPSF30) of the complex that mediates APA and a central transducer of signals initiated by various stimuli – calmodulin – is important for the functioning of CPSF30 in the plant. This hypothesis was confirmed for a subset of plant growth phenotypes, but ruled out for others. These findings confirm that there are connections between cellular signaling and APA, but they also indicate that the control of APA is more complicated than originally proposed. The second hypothesis held that inhibition of the activity of CPSF30 would lead to an identifiable set of changes in APA in the plant. The rationale behind this second hypothesis lies in the finding that calmodulin inhibits the activity of CPSF30. This second hypothesis was confirmed. Specifically, it was found that APA mediated by CPSF30 affects a large number of genes in Arabidopsis (the plant with which this research was conducted), including a disproportionate number that have previously been connected with stress responses in plants. Together, these results show how stimuli lead to a large-scale remodeling of mRNA polyadenylation and thus gene expression in the plant. In terms of the agricultural enterprise, these outcomes reveal one mechanism by which plants respond to a variety of stresses, and thus facilitate rapid adaptation and survival upon the onset of stress. This information will be helpful in devising new approaches for limiting the impacts of disease and changing climate on crop plant productivity. This project provided research opportunities for two graduate students, five undergraduate students (from the University of Kentucky and three other institutions, including an HBCU), a high school teacher, and a visiting scholar from India. In addition, it laid a foundation for extensive outreach and educational activities involving the development of materials and laboratory methods for teaching Next Generation DNA Sequencing in undergraduate laboratories. These are expected, in the future, to reach more than 1000 students across the nation.
