Genome-wide analysis of anterior-posterior mRNA regionalization in Stentor coeruleus reveals a role for the microtubule cytoskeleton
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Abstract
Cells have complex and beautiful structures that are important for their function. However, 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 have 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 membranellar band, 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. 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.
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We do note however, that for a small number of genes, the skew in the control RNAi half-cells was opposite to that seen in bulk RNA sequencing but then became the same as in bulk RNA sequencing in the RNAi cells.
Might it be possible that the specific cells used for the half-cell experiment were in a rare or atypical cell state, which is underrepresented in the bulk data?
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(A) PCA on control half-cell RNA data showing a clear separation of anterior (circles) and posterior (x’s) on PC2.
For ease of comparison to Fig. 1, perhaps you could use the same color scheme to demarcate A/P in the PCA plots, and use shape to indicate RNAi control vs. treatment.
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The sequence targeting the β-tubulin gene, SteCoe_5719 was amplified with the following oligos: forward - 5’ ATGGTGACTTAAATCACTTGGTTAGTGC 3’, reverse – 5’ TTAAGCAGCTTCCTCTTCCTCATCG 3’
To control for possible off-target effects, would it be reasonable to design a separate set of primers?
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SteCoe_37805
Are there candidate gene names associated with these transcripts? For the reader, using those aliases may be easier to understand/remember as opposed to a variety of barcodes.
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This threshold allows for exploratory analysis of more candidate genes in the future, although we acknowledge that a q-value of 0.2 may result in a higher false discovery rate.
How many genes would be considered significantly varying if the q-value was instead 0.05 or 0.01?
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