Cells employ ribosomes and transfer RNA (tRNA) molecules to translate sequence information encoded in mRNAs and produce correctly folded proteins. These molecular machines subsequently carry out most of the enzymatic functions in all living cells and organisms. Regulatory mechanisms concerning this fundamental decoding process involve various post-transcriptional RNA modifications, currently embraced by the term “epitranscriptome”.
The proposed research plan focuses on dissecting molecular mechanisms that allow the highly conserved Elongator complex to conduct specific modifications of uridine bases located in the wobble base position of tRNAs. Its modification activity is essential for maintaining accurate translational rates, protein folding dynamics, global protein homeostasis as well as precluding severe human diseases. In detail, we would like to formulate the following research questions and outline how we plan to answer these points with the proposed research project.
We plan to combine cryo-electron microscopy with detailed mechanistic and functional studies using protein biochemistry, biophysics and in vivo validation. In detail, we plan to obtain high resolution electron densities of the fully assembled Elongator complex bound to endogenous target tRNAs, which are decorated with different combinations of other types of tRNA modifications. Furthermore, we aim to determine the structures of the thiolation cascade components bound to Elongator, which are recruited to the complex via one of its regulatory factors. In addition, we will test the interaction of Elongator with various kinases, analyze the influence of the resulting phosphorylation events and determine the structure of the identified complexes.
We will use complementary protein-protein and protein-tRNA interaction assays in vitro to define stable entities suitable for homogenous vitrification. Vice versa we intend to use these assays for functional validation of our structural information using structure-guided mutational approaches. Obtained mechanistic knowledge will be confirmed with the help of long-term collaboration partners at German and Swiss research institutes by testing functionally relevant mutants in established phenotypical assays in vivo using model organisms like yeast, fish and worms.
Foremost, the expected results from our proposed research plan will provide deep molecular and
functional insights into yet poorly characterized regulatory networks of protein synthesis and proteome homeostasis. Furthermore, gaining insights into the respective cellular mechanisms is also of prime clinical importance as the occurrence of specific mutations in different Elongator subunits in patients is directly correlated with the onset of neurodegenerative diseases and the appearance of treatment resistant cancer cells.
In summary, all investigated protein complexes are highly conserved from yeast to human, execute crucial cellular functions and have strong clinical importance for various severe diseases in humans, making them potentially interesting diagnostic markers and intervention points for future targeted therapies.
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