PROJECT SUMMARY Cells are the building blocks that comprise and pattern multicellular organisms from plants to humans, and many types of cells themselves exhibit complex shapes and patterns. While we understand many of the molecular components of cells, we know much less about how they are assembled to create patterns at the cellular scale. RNA regionalization, or spatially distinct localization of transcripts, is known to contribute to cellular morphology and function in various cells such as neurons. Several diseases that arise in the brain, from depression to Alzheimer’s, may also partly be explained by mislocalized RNA. To understand how cells are patterned via RNA regionalization, we must also understand how the genome is spatially organized as nuclear architecture and chromatin structure both influence gene expression. My proposal leverages the strengths of a unicellular system, Stentor coeruleus, that possesses a complex cellular architecture including a mouth and tail, and is capable of regeneration when damaged. Stentor cells also harbor a transcriptionally active macronucleus that spans the 1 mm long axis of the cell with a unique beads-on-a-string configuration and contains more than 50,000 copies of the genome. These properties enable microsurgery to physically separate regions of the cell and its nucleus along a defined axis for sub-cellular and sub-nuclear analyses of RNA and genome regionalization by sequencing, which is not possible in traditional model systems. First, I will examine two independent models for cellular patterning by RNA regionalization. With RNAi and RNA- sequencing in bisected cells, I will determine whether RNA transport promotes cellular asymmetry by defining the cytoskeletal elements and associated motors required for RNA regionalization. With RNA-sequencing in individual ‘nodes’ of the macronucleus, I will also determine if gene expression is regionalized, thereby reducing the distance RNA must travel before it reaches its desti