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Transfer RNA (tRNA) is crucial for protein synthesis, constituting the adaptor molecule between messenger RNA (mRNA) and protein (Hopper & Phizicky, 2003). Despite its critical function, we still lack key knowledge into the exact mechanisms how tRNA interacts with mRNA and, in particular, the role of chemical RNA modification. A mature tRNA molecule contains numerous chemical modifications of RNA nucleosides (Machnicka et al., 2013), many of which are conserved across broad phylogenetic boundaries (Helm, 2006). These modifications regulate several aspects of translation and affect a wide variety of physiological properties, including temperature tolerance and pathogenicity (Sarin & Leidel, 2014).

The aim of our research is to understand how tRNA modifications in host organisms and microbial pathogens are affected at different stages of infection, and how these changes manifest in translation.

How does host cell translation change upon infection?

Host organisms undergo marked reprogramming of their transcriptome during infection. Common transcriptional activation programmes act as generic alarm signals and induce cytokine expression (Jenner & Young, 2005). The extent at which host transcription is affected depends on the nature of the pathogen. Extracellular infections lead to general transcriptional rearrange-ment of common stress responses, such as heat shock proteins, metabolic stress response, and ubiquitin (Lee & Young, 2013), whereas intracellular infections cause global shut-off of host mRNA translation, which cripples anti-pathogenic responses (Lemaitre & Girardin, 2013; Walsh et al., 2013). However, little is known about how translation is affected during the early stages of infection, particularly how infection changes the tRNA modification levels (Koh & Sarin, 2018). It has recently been shown that physiological stress factors can alter tRNA modification levels (Alings, Sarin et al., 2015). This potentially leads to translational slowdown and upregulation of several stress response pathways, including expression of genes associated with proteotoxic stress (Nedialkova & Leidel, 2015).

Since infection constitutes a severe stress to the host, changes in tRNA modification levels and translation are expected. This hypothesis will be challenged by studying the early stages of infection in well-defined infection models, determining host tRNA modification levels and translation rates, and comparing infected cells to non-infected controls. This will be achieved by the combined use of state-of-the-art techniques, including nano-LC mass spectrometry and ribosome profiling.

REFERENCES:
Alings, F., Sarin, L. P., Fufezan, C., Drexler, H. C., & Leidel, S. A. An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast. RNA 21(2): 202-212 (2015).
Jenner, R. G. and Young, R. A. Insights into host responses against pathogens from transcriptional profiling. Nat. Rev. Microbiol. 3(4): 281-294 (2005).
Koh, C. S. and Sarin, L. P. Transfer RNA modification and infection - Implications for pathogenicity and host responses. BBA Gene Regul. Mech. 1861(4): 419-432 (2018).
Lee, T. I. and Young, R. A. Transcriptional regulation and its misregulation in disease. Cell 152(6): 1237-1251 (2013).
Lemaitre, B. and Girardin, S. E. Translation inhibition and metabolic stress pathways in the host response to bacterial pathogens. Nat. Rev. Microbiol. 11(6): 365-369 (2013).
Nedialkova, D. D. and Leidel, S. A. Optimization of codon translation rates via tRNA modification maintains proteome integrity. Cell 161(7): 1606-1618 (2015).
Walsh, D., Mathews, M. B. and Mohr, I. Tinkering with translation: protein synthesis in virus-infected cells. Cold Spring Harb. Prospect. Biol. 5(1): a012351 (2013).

How do spe­cific tRNA modi­fic­a­tions fa­vour patho­gen­i­city?

Pathogens need to rapidly adapt to continuously changing environmental conditions. Factors that affect microbial virulence include accurate sensing of the environment (quorum sensing), secretion of chemicals into the surroundings (secretion systems), adhering to and colonising of vast areas (biofilm formation), directed motility, and growth at elevated temperatures. These factors are triggered by external signals that indicate poor growth conditions or hostile environments, including high temperature, pH changes, presence or absence of certain nutrients, cell density, and many more (Polke et al., 2015; Koh & Sarin, 2018). A hallmark of opportunistic pathogens is their ability to rapidly switch between commensal and invasive transcription programmes. Their genomes also include hot spots where mutations are frequent, such as the repetitive sequences of minisatellites, telomere regions, and tRNA genes (Koh & Sarin, 2018). Moreover, small regulatory RNAs have been reported to modulate expression of virulence-associated functions in bacteria (Papenfort & Vanderpool, 2015), and thiolation of U34 in three tRNA isoacceptors contributes to high temperature growth and increased virulence in yeast (Koh & Sarin, 2018).

Thiolated tRNA modifications are of particular interest as they modulate temperature tolerance in several species. However, it stands to reason that other tRNA modifica-tions might benefit pathogenicity. To test this theory, tRNA modification and translation will be quantitatively analysed at different stages of infection. Pathogen-specific tRNAs are analysed by nano-LC mass spectrometry, providing information about the basal level of tRNA modification and its changes throughout infection. Thus, over- or under-represented nucleoside modifications are identified and their position within specific isoacceptors can be determined. The effect of such candidate modifications on microbial viability will be assessed by RNA modification enzyme knockout experiments or comple-mentation assays by overexpressing relevant hypomodified tRNA isoacceptors. Finally, translational changes will be determined by ribosome profiling.

REFERENCES:
Papenfort, K. and Vanderpool, C. K. Target activation by regulatory RNAs in bacteria. FEMS Microbiol. Rev. 39(3), 362-378 (2015).
Polke, M., Hube, B. and Jacobsen, I. D. Candida survival strategies. Adv. Appl. Microbiol. 91, 139-235 (2015).
Koh, C. S. and Sarin, L. P. Transfer RNA modifications and infection -- Implications for pathogenicity and host responses. BBA Gene Regul. Mech. 1861(4): 419-432 (2018).

What is the func­tion of virus-en­coded tRNA mo­lecules?

Many obligate cellular parasites utilize different aspects of tRNA biology to facilitate host infection, although the exact mechanism of this regulation is largely unknown. Retroviruses constitute the best-studied example where specific cellular tRNA isoacceptors are hijacked to prime the viral reverse transcriptase enzyme, allowing integration of the viral genome into the host genome (Kelly et al., 2003). Viruses that encode their own limited repertoire of tRNA genes are far less studied, although these include several bacteriophages, nucleoplasmic DNA viruses, positive-strand RNA plant viruses, and gammaherpesviruses infecting humans and other mammals. These virus-encoded tRNAs (vtRNAs) are thought to modulate  translation by supplementing codons that are infrequent in the host but frequent in the viral genome (Dreher, 2010). Previous studies indicate that vtRNAs might be amino-acylated (Cliffe et al., 2008; van Etten & Dunigan, 2012), suggesting that they are properly processed and possibly decorated with chemical modifications.

To elucidate the function of vtRNAs, we will isolate vtRNA molecules from infected host cells and, using a variety of techniques, reveal if the vtRNAs are fully processed and chemically modified, after which the modifications will be mapped onto specific vtRNA isoacceptors. The genome of the virus will be screened for putative RNA modification enzymes, which will be expressed and characterized for enzymatic activity. Furthermore, vtRNA aminoacylation will be elucidated and the capacity of vtRNAs to affect translation rates and alter codon usage will be determined using ribosome profiling. This will address the functional coding capacity of vtRNAs and allow us to correlate possible changes in translation with vtRNA levels in host cells.

REFERENCES:
Cliffe, A. R., Nash, A. A. and Dutia, B. M. Selective uptake of small RNA molecules in the virion of murine gammaherpesvirus 68. J. Virol. 83(5): 2321-2326 (2008).
Dreher, T. W. Viral tRNAs and tRNA-like structures. Wiley Interdiscip. Rev. RNA 1(3): 402-414 (2010).
van Etten, J. L. and Dunigan, D. D. Chloroviruses: not your everyday plant virus. Trends Plant Sci. 17(1): 1-8 (2012).
Kelly, N. J., Palmer, M. T. and Morrow, C. D. Selection of retroviral reverse transcription primer is coordinated with tRNA biogenesis. J. Virol. 77(16): 8695-8701 (2003).

Transfer RNA modification and cancer – tinkering with translation.

Translation is rigorously regulated in healthy cells to control accuracy, rate, and homeostasis during protein synthesis. This careful balance is disrupted in cancer cells, where changes in tRNA modification levels are essential to allow increased protein synthesis and sustained neoplastic proliferation (Sarin & Leidel, 2014). Early studies revealed that tumors have significantly altered tRNA methylation and queuosine modification levels compared to healthy tissue (Kerr & Borek, 1973; Katze & Beck, 1980), although the first direct link between tRNA modification and neoplasia was not established until 2016 (Delaunay et al. 2016). Despite recent advances, the effect of aberrant tRNA modification on tumor formation and proliferation is poorly understood.

To elucidate the translation mechanisms by which tumors are formed, we aim to (i) identify tRNA modifications with significantly altered abundance and (ii) investigate tRNA modification-mediated translational changes in various cancers. We hypothesize that cancer cells can be rescued by pinpointing tRNA modifications that maintain tumorigenic growth and correct their modification levels to that of healthy, non-oncogenic cells. To this end, we use mass spectrometry and ribosome profiling to quantitatively determine tRNA modification levels and monitor translational changes in healthy vs. cancerous cells. The follow-up correlation of RNA modification and translation changes will create a detailed characterization of tRNA-mediated translational responses and identify key regulatory modifications on specific tRNA species.

This study, funded by the Sigrid Jusélius Foundation, will highlight the regulatory function of tRNA modifications and expand our understanding of cancer formation.

REFERENCES:
Delaunay, S., Rapino, F., Tharun, L., Zhou, Z., Heukamp, L, et al. Elp3 links tRNA modification to IRES-dependent translation of LEF1 to sustain metastasis in breast cancer. J. Exp. Med. 213(11): 2503-2523 (2016).
Katze, J.R. and Beck, W. T. Administration of queuine to mice relieves modified nucleoside queuosine deficiency in Erlich ascites tumor tRNA. Biochem. Biophys. Res. Comm. 96(1): 313-319 (1980).
Kerr, S.J. and Borek, E. Regulation of the tRNA methyltransferases in normal and neoplastic tissue. Adv. Enzyme Reg. 11: 63-77 (1972).
Sarin, L. P. and Leidel, S. A. Modify or die? – RNA modification defects in metazoans. RNA Biol. 11(12): 1555-1567 (2014).

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.

REFERENCES:
Alings F, Sarin LP, Fufezan C, Drexler HC, Leidel SA. An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast. RNA 21(2): 202-212 (2015).
Gingold H, Tehler D, Christoffersen NR, Nielsen MM, Asmar F, et al. A dual program for translation regulation in cellular proliferation and differentiation. Cell 158(6): 1281-1292 (2014).
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).
Nedialkova DD, Leidel SA. Optimization of codon translation rates via tRNA modifications maintains proteome integrity. Cell 161(7): 1606-1618 (2015).
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).