Endogenous protein synthesis is regulated at the transcriptional and translational level to yield a balanced proteome that is adapted to the current needs of the cell (Quax et al., 2015). Heterologous protein production is far less straightforward as translational bottlenecks caused by mismatches in codon usage and availability frequently necessitates codon optimization to improve production yields. However, since post-transcriptional tRNA modifications have been shown to influence translation (Laxman et al., 2013; Gingold et al., 2014; Nedialkova & Leidel, 2015), we hypothesize that their dynamic nature (Alings et al., 2015) can be harnessed to fine-tune translation and thus prime the production cells for the altered translational requirements posed by heterologous protein production.
To elucidate the relationship between translational fine-tuning and tRNA modification, we aim to elucidate how: (i) tRNA modifications change upon translation of heterologous gene transcripts, (ii) the tRNA modification profile can be adapted to further heterologous protein production, and (iii) stable these translational optimization measures are during extended production periods. For this purpose, we utilize various fusion protein constructs as reporters in Saccharomyces cerevisiae and Nicotiana tabacum, where changes in tRNA modification levels and translation will be monitored using mass spectrometry (Sarin et al., 2018) and ribosome profiling (Ingolia et al., 2012), respectively.
This Academy of Finland funded research will address how post-transcriptional tRNA modification can be harnessed for increased proficiency and protein yield in a bioproduction setting.
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Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protoc. 7(8): 1534-1550 (2012).
Laxman S, Sutter BM, Wu X, Kumar S, Guo X, Trudgian DC, Mirzaei H, Tu BP. Sulfur amino acids regulate translational capacity and metabolic homeostasis through modulation of tRNA thiolation. Cell 154(2): 416–429 (2013).
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Quax TE, Claassens NJ, Söll D, van der Oost J. Codon bias as a means to fine-tune gene expression. Mol. Cell 59(2): 149-161 (2015).
Sarin LP, Kienast SD, Leufken J, Ross RL, Dziergowska A, Debiec K, Sochacka E, Limbach PA, Fufezan C, Drexler HCA, Leidel SA. Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels. RNA 24(10): 1403-1417 (2018).