Genome-wide analysis of anterior-posterior mRNA localization in Stentor coeruleus reveals a role for the microtubule cytoskeleton

Cells have complex and beautiful structures that are important for their function, but understanding the molecular mechanisms that produce these structures is a challenging problem due to the gap in size scales between molecular interactions and cellular structures. The giant ciliate Stentor coeruleus is a unicellular model organism whose large size, reproducible structure, and ability to heal wounds and regenerate has historically allowed the formation of structure in a single cell to be addressed using methods of experimental embryology. Such studies have shown that specific cellular structures, such as the oral apparatus, always form in specific regions of the cell, which raises the question: what is the source of positional information within this organism? By analogy with embryonic development, in which localized mRNA is often used to mark position, we asked whether position along the anterior-posterior axis of Stentor might be marked by specific regionalized mRNAs. By physically bisecting cells and conducting half-cell RNA sequencing, we were able to identify sets of messages enriched in either the anterior or posterior half. We repeated this analysis in cells in which a set of longitudinal microtubule bundles running down the whole length of the cell, known as KM-fibers, were disrupted by RNAi of b-tubulin. We found that many messages either lost their regionalized distribution or switched to an opposite distribution, such that anterior-enriched messages in control became posterior-enriched in the RNAi cells, or vice versa. This study indicates that mRNA can be regionalized within a single giant cell and that microtubules may play a role, possibly by serving as tracks for the movement of the messages.