Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability

Authors: GORJIFARD, SAYEH - University Of Washington; JORES, TOBIAS - University Of Washington; TONNIES, JACKSON - University Of Washington; MUETH, NICHOLAS - University Of Washington; BUBB, KERRY - University Of Washington; WRIGHTSMAN, TRAVIS - Cornell University; Buckler, Edward - Ed; FIELDS, STANLEY - University Of Washington; CUPERUS, JOSH - University Of Washington; QUEITSCH, CHRISTINE - University Of Washington

Submitted to: Nature Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2024
Publication Date: 7/12/2024
Citation: Gorjifard, S., Jores, T., Tonnies, J., Mueth, N.A., Bubb, K., Wrightsman, T., Buckler Iv, E.S., Fields, S., Cuperus, J.T., Queitsch, C. 2024. Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability. Nature Communications. Vol 15; p.5868. https://doi.org/10.1038/s41467-024-50174-7.
DOI: https://doi.org/10.1038/s41467-024-50174-7

Interpretive Summary: Increased climate variability over the coming decades will require plant breeders to more rapidly select the best varieties for these new conditions. Genome engineering has the potential to quickly produce varieties with specific traits, such as resilience to environmental conditions. Unfortunately, genome engineers need specific locations in the genome to target and crop plants have little to no lists of targets for any given trait. The terminator region of a gene could be a potential region to target because it is known to regulate a portion of a gene’s expression, which in turn controls much of a crop’s performance in the field. However, the entire terminator region is still a big target. By studying the sequence patterns that are important drivers of gene expression within the terminator, the most important sites within the terminator can be identified and used as targets for genome engineering. We created a model to better understand how the DNA sequence at the end of a gene controls its activity. We used this model to create optimized plant gene sequences that could be incorporated into crop plant genomes and potentially accelerate breeding efforts. Having optimized plant gene terminator sequences that are specific to a given species will give genome engineers something to test in modern crop varieties for any trait. Further, scientists can use these new terminator sequences to more finely-tune expression levels of their gene constructs, rather than simply expressing as much as possible.

Technical Abstract: The 3’ end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.