The aim of our research is to obtain a holistic understanding of how tRNA modifications modulate translation and affect cellular functions. To this end, we are interested in:
i) exploring how viruses utilize RNA components to reprogramme host cell translation,
ii) deciphering the translational dysregulation that occurs in cancer cells and how PTMs further this process,
iii) understanding how PTMs contribute to tissue development and aging,
iv) investigating PTMs and virus-encoded RNA components as tools for tweaking translation in cell-free bioproduction systems, and
v) developing methodological and analytical approaches for analyzing translation.
RNA-based regulatory strategies may play essential roles in the viral infection cycle, whether it is to promote viral replication or to interfere with host cell translation and suppress antiviral responses. Lately, three distinct aspects have emerged by which obligate cellular parasites may use tRNA biology to regulate host infection: PTM dynamics, tRNA-derived fragments, and virus-encoded tRNAs.
PTMs are abundant on tRNA; those at the core of the tRNA modulate structural flexibility, whereas the anticodon stem loop (ASL) constitutes a modification hotspot that primarily affects translation rate and fidelity. We have recently shown that tRNA modifications are not static during infection, but they undergo marked changes in abundance throughout the viral replication cycle. We hypothesize that viral infection impacts specific PTMs to further the expression of viral proteins, thus bridging potential translational bottlenecks caused by mismatches in codon usage and tRNA availability.
Some viruses infecting eukaryotic hosts have been reported to suppress host antiviral responses by fragmenting specific host tRNAs. These infection-induced tRNA-derived fragments (tRFs) further viral replication through a poorly understood trans-silencing mechanism. Interestingly, we observe virus-induced fragmentation of a specific host tRNA at the early and mid-infection stage in our prokaryotic host-pathogen model, suggesting that tRFs are evolutionary conserved and possibly confer similar functions.
Many viruses encode their own repertoire of tRNA genes that are poorly studied despite being widespread. Virus-encoded tRNAs (vtRNAs) may supplement codons that are infrequent in the host but frequent in the viral genome. Some vtRNAs are aminoacylated by host tRNA synthetases, suggesting that they may be bona fide translational components. Moreover, aberrantly processed or modified tRNAs are targets for nuclease degradation, implying that vtRNAs may serve as tRF yielding substrates. Since viruses have been evolutionary trimmed to maintain a minimal genome size, it is unlikely that vtRNAs are kept as non-functional ‘junk’. Thus, the exact role of tRFs and vtRNAs in protein synthesis remains to be elucidated.
Aberrant tRNA modification affects protein homeostasis, resulting in proteotoxic stress followed by apoptosis or uncontrolled cellular growth. In humans, this imbalance manifests as severe developmental and neurological dysfunctions, as well as cancers.
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. To elucidate the translation mechanisms by which tumors are formed, we aim to (i) identify PTMs with significantly altered abundance and (ii) investigate tRNA modification-mediated translational changes in various cancers.
We observe that colorectal adenocarcinoma cells undergo marked PTM changes upon metastasis, suggesting that tumor formation and subsequent metastasis undergo translational reprogramming that either is furthered by or necessitates dynamic adaptation of tRNA modification. Remarkably, tRNA modifications at or near the ASL are significantly altered, implicating a direct impact on translation either via altered codon-anticodon interaction and base pairing or via tRNA interaction with the ribosome. Furthermore, 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.
This project, funded by the Sigrid Jusélius Foundation, provides a detailed understanding of the translational impact of tRNA modifications, and enables the enhancement of established treatment regimes.
PTM levels are constantly changing and adapt as the cell – and in a broader context the organism – develops and ages. Conversely, loss of specific PTMs in metazoans has been linked to certain forms of developmental disorders and autosomal-recessive intellectual disabilities. These disorders are attributed to decreased proliferation rates, global translation changes, and perturbations in redox homeostasis. Thus, it is conceivable that aging may be associated with a similar perturbation of proteostasis and reduced stress tolerance. To this end, we study how PTM levels evolve throughout development to determine the impact of aging on translation. Furthermore, this provides insights into tissue specific development and differences in PTM patters.
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. 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 PTMs have been shown to influence translation, we hypothesize that their dynamic nature can be harnessed to optimize translation and thus prime the bioproduction system for the altered translational requirements posed by heterologous protein production.
To establish PTM-based translational optimization, we have developed a Saccharomyces cerevisiae -based cell-free protein synthesis system that has been primed for analyzing the impact of various RNA components on translation. This allows us to verify the effect of individual components on the translation machinery, explore the impact of novel translation components, as well as obtain tuned reactions settings for the optimal production of the desired products.
This Novo Nordisk Foundation funded research will address how post-transcriptional tRNA modification can be harnessed for increasing the proficiency of cell-based bioproduction systems.
We are excited to work in a rapidly progressing field and to contribute to its exploration by developing methodological and analytical approaches for studying translation. To this end, we have
(i) developed a novel UPLC-MS method that radically reduces sample-to-sample runtime while maintaining excellent resolution and baseline separation of >50 PTMs in targeted or global MS analysis applications,
(ii) created a robust direct RT-qPCR method for virus detection sans genome extraction,
(iii) implemented polysome and ribosome profiling workflows for various organisms, and
(iv) we are continuously exploring and developing improvements for RNA-seq based approaches.
These tools provide the analytical pipeline for correlating PTM dynamics and translation changes, uncovering detailed mechanisms of tRNA-mediated translational responses in diverse organisms.