Collaborative Research: Variability in the Plant Polyadenylation Complex

Messenger RNA polyadenylation is a vital step in the expression of nuclear genes in eukaryotes. This step is mediated by a complex (the PolyAdenylation Complex, or PAC) that is widely conserved. However, aspects of the PAC - its composition, organization, and regulation – differ across eukaryotes. As the PAC determines the usage of poly(A) sites (and thus defines the scope of alternative polyadenylation), knowledge of the dynamic nature of the complex is essential for understanding the means by which the PAC is regulated, and thereby impacts growth and development. The plant PAC is distinctive in many ways, and an abundance of past research suggests a remarkable degree of variability and flexibility in the complex. The research described in this proposal will test a model that holds that variability of the plant PAC directs alternative poly(A) site choice, and that this variability is a key mechanism by which the plant PAC is regulated. Intellectual merit Three specific aims are proposed herein. One aim tests the hypothesis that the functional activity of subunits of the PAC varies in specific cells and tissues, and that this variability reflects regulatory interactions of the PAC. The second tests the hypothesis that the composition of the plant PAC determines its RNA substrate specificity in vitro. The third aim tests the hypothesis that the composition of the plant PAC can vary, and that this variability is determined by the presence or absence of core subunits of the complex. These hypotheses will be tested using a diverse range of experimental approaches. Genetic and transgenic approaches will be used to generate Arabidopsis lines that express different PAC subunits. The activity of the PAC will be assayed in different root cells and tissues using single cell transcriptomics approaches. The in vitro activity of the PAC will be assayed in extracts prepared from the nuclei of cells that have different PAC compositions. The compositions of complexes affinity-purified from these different lines will be determined by immunoblotting and mass spectrometry. Together, this battery of approaches will build out a model that connects PAC composition with activity, and with regulated alternative polyadenylation. Broader impacts The proposed research will provide new insights into composition and flexibility of an evolutionarily-conserved complex, and will impact fields of study beyond plant science. These studies will engage two postdoctoral scientists, a graduate student, and several undergraduate trainees. The PIs will host a series of week-long workshops directed towards faculty at undergraduate-focused and minority-serving institutions. In these workshops, participants will be trained in aspects of proteomics, next generation sequencing, and single cell transcriptomics. These workshops will provide opportunities for participants to enhance their research and teaching portfolios.