Study on preferential translation under pronounced translation
repression caused by brewing-related stress in Saccharomyces
cerevisiae
Shingo Izawa
Faculty of Applied Biology, Graduate School of Science and Technology,
Kyoto Institute of Technology
Report: We have investigated RNA-binding proteins (RBPs) involved in preferential
translation under pronounced translation repression caused by severe ethanol and vanillin
stress in Saccharomyces cerevisiae. We have succeeded to identify several factors thatinteract with the RBPs and other mRNAs that may be effectively translated under the tested
stress conditions.
Research aims
Yeast cells are exposed to various kinds of stress during the process of alcoholic
fermentation. Ethanol, which is produced by yeast, causes severe repression of overall
protein synthesis in yeast cells at high concentrations (>9% v/v). Representative
fermentation inhibitors derived from lignocellulosic biomass such as vanillin, furfural, and5-hydroxymethylfurfural (HMF) also cause pronounced translation repression and preventthe efficient and economical production of second-generation bioethanol1).
Recently, Zid and O’Shea found that HSP26 and HSP30 mRNAs are selectively andefficiently translated despite the pronounced translation repression caused by glucose
depletion, suggesting that crucial mRNAs that are essential for survival under stressconditions can be preferentially translated2). We have also reported that the BTN2 mRNAcan be preferentially translated under severe ethanol stress and ADH7 and BDH2 mRNAs
are efficiently translated under severe vanillin stress1, 3, 4). Very intriguingly, suchpreferential and selective translation under pronounced translation repression is enabled by
DNA promoter regions but not by mRNA sequences1-4). Therefore, it is considered thatRNA-binding proteins (RBPs) are involved in the preferential translation. In order to get aclue on the mechanisms underlying preferential translation, we identified the RBPs that
respond to severe ethanol and vanillin stress and carefully examined them in this study.
Methods
In order to identify key factors for the preferential translation under severe ethanol andvanillin stress conditions, expression of BTN2 and ADH7 in RBP-deficient mutants (205strains) was examined by qPCR and western blot analyses. Proteins and mRNAs that interact
with the RBPs upon stress were identified by co-immunoprecipitation (co-IP) and DNA
microarrays.
Results
Through the careful examination of 205 RBP-deficient mutants, we identified 7 RBPs
involved in the efficient protein synthesis of Btn2 or Adh7 under pronounced translation
repression caused by severe ethanol or vanillin stress. Although the deficient mutants ofthese RBPs showed almost identical levels of mRNA synthesis in the wild type, the protein
synthesis of Btn2 and Adh7 was severely repressed. rbp26∆ cells hardly induced Bnt2
protein synthesis under 10% ethanol and showed hypersensitivity to ethanol stress. Co-IP
analysis indicated that Rbp26 interacted with Hsp70s and translation elongation factors(eEFs) in the presence of 10% ethanol. It is known that the mammalian eEF-1A moves to thenucleus from the cytoplasm and then escorts HSP mRNAs to the translation machinery for
efficient translation under heat shock conditions5). We currently speculate that yeast cells
might have a similar system that facilitates translation under severe ethanol stress using eEFs.
We also analyzed mRNAs bound to Rbp26 in the presence of 10% ethanol, as they arecandidates that can be efficiently translated under severe ethanol stress. We are now
verifying for their preferential translation under 10% ethanol stress.
Rbp15 seems to be critical for the efficient translation of ADH7 under severe vanillin
stress, and a similar analysis to that performed for Rbp26 was carried out. We found that
YLL056c mRNA bound to Rbp15 in the presence of vanillin. Although the expression ofYLL056c was strongly repressed at the transcriptional level under non-stress conditions, it
was significantly activated not only at the transcriptional level but also at the translational
level under severe vanillin stress6). The induced expression levels of YLL056c weremarkedly higher than those of ADH7 and BDH2 upon vanillin stress. Expression of YLL056c
was also induced by furfural and HMF, and the yll056c∆ cells presented hypersensitivity tovanillin, furfural, and HMF. The YLL056c promoter could induce the efficient expression ofother non-related genes such as ALD6 and GFP under pronounced translation repression
caused by vanillin, furfural, or HMF. Since the gene expression under the control of theYLL056c promoter was not induced under non-stress conditions, the YLL056c promoter
seems to have an advantage over other constitutive strong promoters to reduce the burden on
yeast cells after a reduction in vanillin concentration. We think that the VFH1 promoter
constitutes a useful tool for improving the stress tolerance to vanillin, furfural, and HMF6).

Conclusion
Our data suggest that Rbp26 facilitates the preferential translation of BTN2 mRNA via theformation of a complex with Hsp70s and eEF under severe ethanol stress conditions. By
analyzing Rbp26-bound mRNAs, we also identified other candidates that can bepreferentially translated in the presence of 10% ethanol. Regarding the response to severe
vanillin stress, we found that the YLL056c promoter is able to modify gene expression in thepresence of fermentation inhibitors derived from lignocellulosic biomass. There is strong
indication for the superior utility of the YLL056c promoter for breeding optimized yeast
strains in bioethanol production.
References
1) Kato, S., and Izawa, S. (2018) Improvement of yeast fermentation efficiency utilizingmRNAs preferentially translated under translational repression. in Applied RNA
Bioscience. Springer. ISBN 978-981-10-8371-6.
2) Zid, B. M., and O'Shea, E. K. (2014) Promoter sequences direct cytoplasmic localizationand translation of mRNAs during starvation in yeast. Nature, 514:117–121.
3) Yamauchi, Y., and Izawa, S. (2016) Prioritized expression of BTN2 of Saccharomyces
cerevisiae under pronounced translation repression induced by severe ethanol stress.
Front. Microbiol. 7:1319.
4) Kato, S., Yamauchi, Y., and Izawa, S. (2018) Protein synthesis of Btn2 underpronounced translation repression during the process of alcoholic fermentation andwine-making in yeast. Appl. Microbiol. Biotechnol., 102:9669–9677.
5) Vera, M. et al. (2014) The translation elongation factor eEF1A1 couples transcription totranslation during heat shock response. eLife 3:e03164. doi: 10.75554/eLife.03164
6) Nguyen, T. T. M., Ishida, Y., Kato, S., Iwaki, A, and Izawa, S. (2018) The VFH1
(YLL056C) promoter is vanillin-inducible and enables mRNA translation despite
pronounced translation repression caused by severe vanillin stress in Saccharomyces
cerevisiae. Yeast 35: 465-475.