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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, Pena, Noah, Assari, Mahdi, del Carmen Sánchez Olmos, María, Zhao, Yu, Ballerini, Alba, Blount, Benjamin A., Cai, Jitong, Ogunlana, Lois, Liu, Wei, Jönsson, Katarina, Abramczyk, Dariusz, Garcia-Ruiz, Eva, Turowski, Tomasz W., Swidah, Reem, Ellis, Tom, Pan, Tao, Antequera, Francisco, Shen, Yue, Nieduszynski, Conrad A. 
ORCID: https://orcid.org/0000-0003-2001-076X, Koszul, Romain, Dai, Junbiao, Steinmetz, Lars M., Boeke, Jef D. and Cai, Yizhi
(2023)
Design, construction, and functional characterization of a tRNA neochromosome in yeast.
Cell, 186 (24).
5237-5253.e22.
ISSN 0092-8674
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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 incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, 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 opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
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| Additional Information: | Acknowledgments: This work was supported by UK Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/M005690/1, BB/P02114X/1, and BB/W014483/1; a Volkswagen Foundation “Life? Initiative” Grant (Ref. 94 771); an Engineering and Physical Sciences Research Council (EPSRC) Fellowship EP/V05967X/1; and a European Research Council (ERC) Consolidator Award EP/Y024753/1 to Y.C. D. Schindler is supported by the Max Planck Society in the framework of the MaxGENESYS project. R.S.K.W., at the University of Edinburgh, was supported by EPSRC studentship (1419736) and the Bill and Melinda Gates Foundation (OPP1140908) to Y.C. R.S.K.W., as part the Yeast 2.0 initiative at Macquarie University, is financially supported by Bioplatforms Australia, the New South Wales (NSW) Chief Scientist and Engineer, and the Australian Research Council (ARC) Centre of Excellence in Synthetic Biology. N.P. is supported by NIH/R01GM113194 supplement. M.A. and T.P. are supported by NIH/NHGRI RM1HG012780. M.d.C.S.O. is supported by the International Max Planck Research School for Principles of Microbial Life: from molecules to cells, from cells to interactions. F.A. is supported by a grant from the Spanish Ministry of Science and Innovation (PID2020-118423GB-I00). C.A.N. acknowledges support from the Biotechnology and Biological Sciences Research Council (BBSRC), part of UK Research and Innovation, grant BB/N016858/1; Core Capability Grant BB/CCG1720/1; and the National Capability (BBS/E/T/000PR9814). J.D. is supported by a Royal Society Newton Advanced Fellowship (NAF\R2\180590) hosted by Y.C. J.D.B. was supported by NSF grants MCB-1026068, MCB-1443299, MCB-1616111, and MCB-1921641. We thank Anita Dornes, Patricia Bedrunka, and Gert Bange for using their equipment and materials for western blot analysis. |
| Faculty \ School: | Faculty of Science > School of Biological Sciences |
| Depositing User: | LivePure Connector |
| Date Deposited: | 16 Nov 2023 02:27 |
| Last Modified: | 24 Nov 2023 02:19 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/93644 |
| DOI: | 10.1016/j.cell.2023.10.015 |
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