Study of differential tRNA post-transcriptional modifications as a mechanism to control proteome composition

At the heart of every organism lies a fundamental component, the cell. Cells are autonomous units capable of growing, dividing, and responding to external and internal stimuli. To achieve these complex tasks they have to tightly coordinate the production and degradation of thousands of proteins, the entirety of which is referred to as the proteome. Protein synthesis starts with the processes of transcribing a "blueprint" - the information in our genes - into an intermediary molecule called messenger-RNA (mRNA). mRNA stores information as a sequence of nucleotide triplets called codons. This information is then translated into a protein. Transfer-RNA (tRNA) molecules play a central role in this process. Different tRNA molecules can recognize different type of codons and translate them into the appropriate amino acids - the building blocks of proteins. The main mechanism by which cells can control the amount of proteins synthesized at any given time, relies on changing the abundance of the corresponding mRNAs.
Interestingly, we recently acquired data pointing to the existence of another, novel mechanism by which cells can control the composition of their proteomes. Namely, we have observed that the efficiency with which tRNA molecules can translate a subset of codons, changes in response to changes in the growth conditions a cell is exposed to. We now propose follow up experiments that will shed light on the mechanisms responsible for regulating the efficiency of codon recognition by tRNA in response to changes in the environment. Furthermore, by assessing which part of the proteome is differentially translated as a consequence of the altered tRNA translation efficiency, the proposed experiments will put our initial observation in a functional context. Ultimately, we expect that the experiments we propose will significantly further our knowledge of a truly fundamental biological process and prove / disprove our hypothesis that modulation of tRNA efficiency is a novel regulatory mechanism for controlling proteome composition.
Miss-regulation of a proteome composition and activity state is intimately connected to the majority of all known human diseases. It is therefore easy to see why gaining an in-depth understanding of the mechanisms used to control protein translation is of such cardinal importance. If our hypothesis proves correct, it would, in the long term, provide an entirely new angle and set of putative targets for drug development. On the other hand, it should also be stressed that deficiencies in the tRNA modifications we propose to study have also been associated, amongst others, with neurological diseases, and decreased tolerance to oxidative stress. Finally, it is interesting to note that one of the modified tRNA molecules we propose to study, and not the unmodified version of this tRNA, is essential for HIV infection. In conclusion, we expect that our research will not only shed light on one of the fundamental processes inside cells, but also contribute to improving the quality of life, wellbeing, and health of our society in the medium term future.

Nucleotides in tRNA molecules are heavily post-transcriptionally modified. Particularly, the first position of the anticodon (i.e. position 34), together with the 3' position adjacent to the anticodon (i.e. position 37), manifest the greatest nucleotide diversity. Although the pathways responsible for the generation of these modified nucleotides are well characterized, the biological function of the modifications often remains elusive. We have recently used a combination of quantitative mass spectrometry and unsupervised machine learning to demonstrate that the thiolation of wobble uridines (s2U34) is important in vivo for the efficient translation of mRNAs rich in AAA, CAA, AAG, and GAA codons. We have since obtained preliminary data showing that the extent of s2U34 modification in tRNAs is regulated in response to growth conditions. Hence, we speculate that differential modification of tRNAs could provide a novel mechanism for fine-tuning the composition of a proteome, by selectively reducing the translation rate of a subset of proteins. Here we propose to further investigate our preliminary findings by identifying a comprehensive set of conditions that results in differential s2U34 modification and by characterizing the molecular mechanisms behind this process. We also propose to probe if all tRNAs which are normally modified by s2U34, or only a subset thereof, are similarly affected under these growth conditions. Since nucleotides modified to s2U34 also bear a second modification, 5-methoxy-carbonyl-methyl (mcm5U34), to generate (mcm5s2U34), and since the two modifications have been previously shown to affect each other, we will also monitor changes in the mcm5U34 modification levels. Finally, we will use this information to predict and validate the proteome subset differentially translated as a direct consequence of the differential tRNA modification.

If financed, our proposal would generate results that would benefit both the basic and the applied fields of science.

Impact on the economy and the pharmaceutical industry:
In the shorter term, the results from this study could provide new mechanistic insights and putative drug targets, for diseases that derive from improper protein translation following deficiencies in tRNA modification (e.g. familial dysautonomia, MERRF, MELAS...) or that depend on tRNA modifications (e.g. lentiviruses, HIV). Importantly, deficiencies in one of the post-transcriptional modifications we propose to study (i.e. wobble uridine thiolation) have also been associated with increased sensitivity to oxidative stress and are therefore of interest to a plethora of human diseases and the process of aging.
The proposed research revolves around preliminary findings pointing to the existence of a novel cellular mechanism to control proteome composition and activity. In the past, findings of such regulatory mechanisms have had a major impact on our health and economic welfare. For comparison terms, the current world market for inhibitors of the kinases - a class of key enzymes responsible for controlling proteome activity and composition - is 18 billion USD per annum and hundreds such molecules are in active clinical development. Therefore, we envisage that if our preliminary results are confirmed, in the long term they could, have a cardinal impact on our health and wellbeing, as well as being a major economic driver.

Technological impact:
In recent years, with the advent of deep-sequencing methodologies, it has become easier than ever to obtain sequence information on DNA/RNA molecules. These methodologies have taken us one step closer to the goal of developing personalized and predictive medicine by enabling the sequencing of individual genomes for a fraction of the time and cost that it would have taken just a few years ago. A facility, equipped with state of the art instrumentation for next-generation sequencing, will in fact soon be established at the College of Life Sciences (CLS) in Dundee. However, an important aspect for the proper function of a DNA/RNA molecule is the post-transcriptional modification of its constituent nucleotides. This process extends the diversity of the 4 canonical bases to a total of more than 100 currently known variants. Unfortunately, next-generation sequencing approaches are unable to measure this type of information and indeed only very few laboratories worldwide are equipped with workflows that allow measurement of these properties. The work proposed in this application, particularly milestone 1 of specific aim 2, would allow the establishment of a pipeline for the modification-aware MS analysis of RNA molecules. This would greatly increase the world competitiveness of the UK in the burgeoning field of DNA/RNA sequencing. Importantly, such a pipeline, would also synergize particularly well with the soon to be next-generation sequencing facility at the Dundee CLS, and help making the college a center of excellence in this field. Overall, we expect this to have a positive economic impact by attracting new researchers and fostering collaborations both within academia and the pharmaceutical industry.