Design, construction, and functional characterization of a tRNA neochromosome in yeast

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

doi:10.1016/j.cell.2023.10.015

Design, construction, and functional characterization of a tRNA neochromosome in yeast

Daniel Schindler, Roy S. K. Walker, Shuangying Jiang, Aaron N. Brooks, Yun Wang, Carolin A. Müller, Charlotte Cockram, Yisha Luo, Alicia García, Daniel Schraivogel, Julien Mozziconacci, Noah Pena, Mahdi Assari, María del Carmen Sánchez Olmos, Yu Zhao, Alba Ballerini, Benjamin A. Blount, Jitong Cai, Lois Ogunlana, Wei LiuKatarina Jönsson, Dariusz Abramczyk, Eva Garcia-Ruiz, Tomasz W. Turowski, Reem Swidah, Tom Ellis, Tao Pan, Francisco Antequera, Yue Shen, Conrad A. Nieduszynski, Romain Koszul, Junbiao Dai, Lars M. Steinmetz, Jef D. Boeke, Yizhi Cai

School of Biological Sciences

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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.

Original language	English
Pages (from-to)	5237-5253.e22
Number of pages	17
Journal	Cell
Volume	186
Issue number	24
Early online date	8 Nov 2023
DOIs	https://doi.org/10.1016/j.cell.2023.10.015
Publication status	Published - 22 Nov 2023

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Schindler, D., Walker, R. S. K., Jiang, S., Brooks, A. N., Wang, Y., Müller, C. A., Cockram, C., Luo, Y., García, A., Schraivogel, D., Mozziconacci, J., Pena, N., Assari, M., del Carmen Sánchez Olmos, M., Zhao, Y., Ballerini, A., Blount, B. A., Cai, J., Ogunlana, L., ... Cai, Y. (2023). Design, construction, and functional characterization of a tRNA neochromosome in yeast. Cell, 186(24), 5237-5253.e22. https://doi.org/10.1016/j.cell.2023.10.015

@article{cf228ca34d7d43ec8ba95b846cb1db64,

title = "Design, construction, and functional characterization of a tRNA neochromosome in yeast",

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.",

author = "Daniel Schindler and Walker, {Roy S. K.} and Shuangying Jiang and Brooks, {Aaron N.} and Yun Wang and M{\"u}ller, {Carolin A.} and Charlotte Cockram and Yisha Luo and Alicia Garc{\'i}a and Daniel Schraivogel and Julien Mozziconacci and Noah Pena and Mahdi Assari and {del Carmen S{\'a}nchez Olmos}, Mar{\'i}a and Yu Zhao and Alba Ballerini and Blount, {Benjamin A.} and Jitong Cai and Lois Ogunlana and Wei Liu and Katarina J{\"o}nsson and Dariusz Abramczyk and Eva Garcia-Ruiz and Turowski, {Tomasz W.} and Reem Swidah and Tom Ellis and Tao Pan and Francisco Antequera and Yue Shen and Nieduszynski, {Conrad A.} and Romain Koszul and Junbiao Dai and Steinmetz, {Lars M.} and Boeke, {Jef D.} and Yizhi Cai",

note = "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.",

year = "2023",

month = nov,

day = "22",

doi = "10.1016/j.cell.2023.10.015",

language = "English",

volume = "186",

pages = "5237--5253.e22",

journal = "Cell",

issn = "0092-8674",

publisher = "Cell Press",

number = "24",

}

Schindler, D, Walker, RSK, Jiang, S, Brooks, AN, Wang, Y, Müller, CA, Cockram, C, Luo, Y, García, A, Schraivogel, D, Mozziconacci, J, Pena, N, Assari, M, del Carmen Sánchez Olmos, M, Zhao, Y, Ballerini, A, Blount, BA, Cai, J, Ogunlana, L, Liu, W, Jönsson, K, Abramczyk, D, Garcia-Ruiz, E, Turowski, TW, Swidah, R, Ellis, T, Pan, T, Antequera, F, Shen, Y, Nieduszynski, CA, Koszul, R, Dai, J, Steinmetz, LM, Boeke, JD & Cai, Y 2023, 'Design, construction, and functional characterization of a tRNA neochromosome in yeast', Cell, vol. 186, no. 24, pp. 5237-5253.e22. https://doi.org/10.1016/j.cell.2023.10.015

TY - JOUR

T1 - Design, construction, and functional characterization of a tRNA neochromosome in yeast

AU - Schindler, Daniel

AU - Walker, Roy S. K.

AU - Jiang, Shuangying

AU - Brooks, Aaron N.

AU - Wang, Yun

AU - Müller, Carolin A.

AU - Cockram, Charlotte

AU - Luo, Yisha

AU - García, Alicia

AU - Schraivogel, Daniel

AU - Mozziconacci, Julien

AU - Pena, Noah

AU - Assari, Mahdi

AU - del Carmen Sánchez Olmos, María

AU - Zhao, Yu

AU - Ballerini, Alba

AU - Blount, Benjamin A.

AU - Cai, Jitong

AU - Ogunlana, Lois

AU - Liu, Wei

AU - Jönsson, Katarina

AU - Abramczyk, Dariusz

AU - Garcia-Ruiz, Eva

AU - Turowski, Tomasz W.

AU - Swidah, Reem

AU - Ellis, Tom

AU - Pan, Tao

AU - Antequera, Francisco

AU - Shen, Yue

AU - Nieduszynski, Conrad A.

AU - Koszul, Romain

AU - Dai, Junbiao

AU - Steinmetz, Lars M.

AU - Boeke, Jef D.

AU - Cai, Yizhi

N1 - 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.

PY - 2023/11/22

Y1 - 2023/11/22

N2 - 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.

AB - 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.

U2 - 10.1016/j.cell.2023.10.015

DO - 10.1016/j.cell.2023.10.015

M3 - Article

VL - 186

SP - 5237-5253.e22

JO - Cell

JF - Cell

SN - 0092-8674

IS - 24

ER -