Collaborative Research: Variability in the Plant Polyadenylation Complex

University Of Kentucky Research Foundation, Lexington KY

Investigators

Abstract

Plants respond to environmental and developmental cues in numerous ways. One such response involves the control of gene expression via a process known as alternative polyadenylation (APA). APA is known to impact plant development and to affect the responses of plants to conditions such as elevated temperature or infection by pathogens. However, many questions remain concerning the connections between the cue (developmental or stress) and the protein machinery (the polyadenylation complex) that mediates and regulates APA. This project focuses on where and when in the plant these connections are made, and how these connections affect the composition of the machinery that mediates the process. To this end, a spectrum of tools will be brought to bear – the activity of the complex will be assayed at the single cell and sub-cellular levels, and the composition of this complex will be studied in mutants of the model plant Arabidopsis that lack strategic protein subunits. This research will lead to a better understanding of the growth and development of crop plants, and their responses to various stresses, the results of which will benefit crop breeding in the future. This project will also provide training opportunities for postdoctoral scientists, undergraduate students, and faculty from a range of post-secondary institutions. Eukaryotic messenger RNA polyadenylation is mediated by a large complex (the PolyAdenylation Complex, or PAC). This project will test the model that variability in subunit composition is a key mechanism by which the plant PAC is regulated. Three aims will be pursued. One will test the hypothesis that the activity of the PAC varies in specific cells and tissues. The second will test the hypothesis that the composition of the plant PAC determines its RNA substrate specificity. The third will test the hypothesis that the composition of the functional plant PAC can vary. A diverse range of experimental approaches will be used. The activity of the PAC will be assayed in different root cells using single cell transcriptomics approaches. The RNA specificity of the PAC will be assayed in nuclear extracts from cells lacking PAC subunits. The compositions of complexes affinity-purified from different mutants will be determined by mass spectrometry. This research will provide new insights into an evolutionarily-conserved complex and will impact fields of study beyond plant science. These studies will engage postdoctoral scientists, graduate students, and several undergraduate trainees. Workshops directed towards faculty at undergraduate-focused and minority-serving institutions will be conducted. Participants will be trained in aspects of proteomics, next generation sequencing, and single cell transcriptomics. 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.