RNA and genome regionalization in giant single cells: implications for cellular patterning

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 destination. Then, with chromatin profiling and
whole-genome sequencing, I will determine whether the genome is regionalized by spatial chromatin regulation
or differential localization of genes themselves and establish whether these are associated with RNA
regionalization and ultimately cellular patterning. To conduct this work, I will receive training in cell biology and
imaging, chromatin biology and genomics, as well as in tool development in emerging model organisms. This
innovative work will serve as the foundation for my independent research program, which aims to advance our
understanding of the consequences of disrupted cellular patterning in development and disease.

Grant Number: 5K99GM154059-02
NIH Institute/Center: NIH
Principal Investigator: Ashley Albright