Intellectual Merit: Regulation of gene expression is critical to all cellular processes, from development to environmental response. One way to silence gene expression is by chromatin modifications that prevent transcription. Such transcriptional gene silencing can be induced by small RNAs in association with Argonaute proteins. This presents a conundrum, because by definition, chromatin modification must occur in the nucleus, but many Argonaute proteins are found in the cytoplasm. The role of cytoplasmic accumulation of nuclear-acting Argonaute proteins is unclear, but might reflect regulation of Argonaute action, or relate to transport of small RNAs between cells. This project will exploit the model plant Arabidopsis thaliana to understand the molecular mechanisms that regulate the subcellular localization of small RNA-mediated transcriptional gene silencing, in particular how ARGONAUTE 4 and RNA Polymerase IV-dependent small RNAs are spatially distributed within a cell and how this relates to their functions within and between cells. Because the molecular function of small RNAs and Argonautes are conserved in many species, investigating the localization of ARGONAUTE 4 at the protein, cellular, and organismal levels, this research will create mechanistic paradigms that could be applicable to gene silencing in many species.
Broader Impacts: This research offers outstanding training for a graduate student and a postdoctoral fellow, who will utilize advanced genomic and bioinformatics tools as well as traditional molecular and genetic analyses. An undergraduate student will also be included in the research environment, simultaneously advancing his or her own education and creating mentoring opportunities for the graduate student and postdoctoral fellow. This research will also make fundamental contributions to our understanding of transcriptional gene silencing, an important aspect of gene regulation and a powerful avenue for crop improvement.