Barcode sequencing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast
Authors:Catalina A. Romila1,#, StJohn Townsend1,2,#, Michal Malecki1,3, Stephan Kamrad1,2,4, María Rodríguez-López1, Olivia Hillson1, Cristina Cotobal1, Markus Ralser2,5 and Jürg Bähler1
doi: 10.15698/mic2021.07.754
Volume 8, pp. 146 to 160, published 10/05/2021.
1 Institute of Healthy Ageing and Department of Genetics, Evolution & Environment, University College London, London WC1E 6BT, UK.
2 The Francis Crick Institute, Molecular Biology of Metabolism Laboratory, London, NW1 1AT, UK.
3 Current address: Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Poland.
4 Current address: Charité Universitätsmedizin Berlin, Department of Biochemistry, Germany.
5 Charité Universitätsmedizin Berlin, Department of Biochemistry, Germany.
# These authors contributed equally.
Keywords:
cellular aging, longevity, parallel genetic screen, competitive growth, lifespan mutants, Bar-seq, S. pombe.
Corresponding Author(s):
Conflict of interest statement:
The authors declare to have no conflicts of interest.
Please cite this article as:
Catalina A. Romila, StJohn Townsend, Michal Malecki, Stephan Kamrad, María Rodríguez-López, Olivia Hillson, Cristina Cotobal, Markus Ralser and Jürg Bähler (2021). Barcode sequenc-ing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast. Microbial Cell 8(7): 146-160. doi: 10.15698/mic2021.07.754
© 2021 Romila et al. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduc-tion in any medium, provided the original author and source are acknowledged.
Abstract:
Ageing-related processes are largely conserved, with simple organisms remaining the main platform to discover and dissect new ageing-associated genes. Yeasts provide potent model systems to study cellular ageing owing their amenability to systematic functional assays under controlled conditions. Even with yeast cells, however, ageing assays can be laborious and resource-intensive. Here we present improved experimental and computational methods to study chronological lifespan in Schizosaccharomyces pombe. We decoded the barcodes for 3206 mutants of the latest gene-deletion library, enabling the parallel profiling of ~700 additional mutants compared to previous screens. We then applied a refined method of barcode sequencing (Bar-seq), addressing technical and statistical issues raised by persisting DNA in dead cells and sampling bottlenecks in aged cultures, to screen for mutants showing altered lifespan during stationary phase. This screen identified 341 long-lived mutants and 1246 short-lived mutants which point to many previously unknown ageing-associated genes, including 46 conserved but entirely uncharacterized genes. The ageing-associated genes showed coherent enrichments in processes also associated with human ageing, particularly with respect to ageing in non-proliferative brain cells. We also developed an automated colony-forming unit assay to facilitate medium- to high-throughput chronological-lifespan studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay. This study provides an effective methodological platform and identifies many new ageing-associated genes as a framework for analysing cellular ageing in yeast and beyond.