Intellectual Merit: Among the many ways that genes are regulated in eukaryotes is via a process termed as alternative polyadenylation; this process entails the differential choice of sites along a primary RNA transcript at which the messenger RNAs will end (and to which the canonical poly(A) tail will be added). Previous NSF-supported research resulted in the discovery of a novel class of alternative polyadenylation sites that are prevalent in genes associated with stress responses in plants. These sites are predicted to reflect novel gene expression and regulatory mechanisms. To test this prediction, the formation and properties of mRNAs that end at these sites will be studied. The hypotheses to be tested in this project are: mRNAs that end at these novel sites are formed by canonical mechanisms of polyadenylation; mRNAs that end at these sites alter the functionalities of the associated mRNAs; and polyadenylation at these sites is required for the functions of genes that possess such sites. This research will involve biochemical assays, high throughput sequencing of DNA tags that query mRNA-poly(A) junctions as well as the positions of RNA polymerase II along the genome, transient gene expression studies in Nicotiana benthamiana, and experiments utilizing Arabidopsis mutants and transgenic plants.
Broader Impacts: The proposed research addresses a new mode of regulation of genes involved in responses of plants to stress. The knowledge gained from these studies will apply to most crop plants, and it is reasonable to expect that new strategies for managing disease and abiotic stress in crop plants will be forthcoming. The research involves a wide range of approaches; trainees will thus gain first-hand experience in biochemistry, genomics and computational biology, and molecular genetics. The research methods and results will be integrated into graduate and undergraduate classes at the University of Kentucky. Additionally, the methods and results will be made available to faculty at a number of undergraduate-focused institutions as part of outreach efforts to develop curricular modules for high throughput DNA sequencing and genomics.
