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. Specifically, the following questions are addressed:
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. 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.
Alings, F., Sarin, L.P., Fufezan, C., Drexler, H.C., & Leidel, S.A. RNA 21, 202-212 (2015).
Nedialkova, D.D. & Leidel, S.A. Cell 161, 1606-1618 (2015).
Jenner, R.G. & Young, R.A. Nat. Rev. Microbiol. 3, 281-294 (2005).
Lee, T.I. & Young, R.A. Cell 152, 1237-1251 (2013).
Lemaitre, B. & Girardin, S.E. Nat. Rev. Microbiol. 11, 365-369 (2013).
Walsh, D., Mathews, M.B., & Mohr, I. Cold Spring Harb. Prospect. Biol. 5, 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). 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. Moreover, small regulatory RNAs have been reported to modulate expression of virulence-associated functions in bacteria (Papenfort & Vanderpool, 2015). Ongoing work suggests that thiolation of U34 in three tRNA isoacceptors contributes to high temperature growth and increased virulence in yeast (manuscript in preparation).
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.
Papenfort, K. & Vanderpool, C.K. FEMS Microbiol. Rev. 39, 362-378 (2015).
Polke, M., Hube, B., & Jacobsen, I.D. Adv. Appl. Microbiol. 91, 139-235 (2015).
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.
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Dreher, T.W. Wiley Interdiscip. Rev. RNA 1, 402-414 (2010).
van Etten, J.L. & Dunigan, D.D. Trends Plant Sci. 17, 1-8 (2012).
Kelly, N.J., Palmer, M.T., & Morrow, C. D. J. Virol. 77, 8695-8701 (2003).