Genome-wide analysis of mRNA regionalization in a giant single cell

The molecular mechanisms that produce cell structure are poorly understood. The giant ciliate Stentor coeruleus is a unicellular model organism whose large size, reproducible structure, and ability to heal wounds and regenerate allow the formation of structure in a single cell to be addressed using methods of experimental embryology. Such studies have shown that specific cellular structures always form in particular 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 regionalized mRNA is often used to mark position, we asked whether specific regionalized mRNAs might mark position along the anterior-posterior axis of Stentor. By physically bisecting cells and conducting bulk RNA sequencing, we were able to identify sets of messages enriched in either the anterior or posterior half and show that RNAi-mediated knockdown of posterior-enriched transcripts corresponding to MYB genes results in a cell's inability to regenerate the posterior portion of the cell body. We then conducted half-cell RNA-sequencing in paired anteriors and posteriors of cells in which the microtubule cytoskeleton was disrupted by RNAi of β-tubulin or dynein intermediate chains. 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.