Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Schindler, Daniel; Walker, Roy S.K.; Jiang, Shuangying; Brooks, Aaron N.; Wang, Yun; Müller, Carolin A.; Cockram, Charlotte; Luo, Yisha; García, Alicia; Schraivogel, Daniel; Mozziconacci, Julien; Blount, Benjamin A.; Cai, Jitong; Ogunlana, Lois; Liu, Wei; Jönsson, Katarina; Abramczyk, Dariusz; Garcia-Ruiz, Eva; Turowski, Tomasz W.; Swidah, Reem; Ellis, Tom; Antequera, Francisco; Shen, Yue; Nieduszynski, Conrad A.; Koszul, Romain; Dai, Junbiao; Steinmetz, Lars M.; Boeke, Jef D.; Cai, Yizhi

Abstract

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Citation

Schindler, D., Walker, R. S., Jiang, S., Brooks, A. N., Wang, Y., Müller, C. A., Cockram, C., Luo, Y., García, A., Schraivogel, D., Mozziconacci, J., Blount, B. A., Cai, J., Ogunlana, L., Liu, W., Jönsson, K., Abramczyk, D., Garcia-Ruiz, E., Turowski, T. W., Swidah, R., …Cai, Y. Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

• Working Paper Type: Working Paper
• Publicly Available Date: Oct 14, 2022
• Public URL: https://nottingham-repository.worktribe.com/output/12036012
• Publisher URL: https://www.biorxiv.org/content/10.1101/2022.10.03.510608v1
• Additional Information: This bioRxiv article is a preprint and has not been certified by peer review.