NCN OPUS 16 2019 – 2023

NCN OPUS 16 2019 – 2023

Molecular Characterization of large tRNA modification Complexes

Glatt-lab

What is the project about?

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.

  1. How are endogenous tRNAs recognized by the Elongator complex and do co-existing modifications in other positions of the tRNA influence the selection of substrate tRNAs?
  2. How does Kti11 recruit the thiolation cascade component Ncs6 to Elongator and does the final sulfur transfer step occur while Ncs6 is still directly associated with Elongator?
  3. How does phosphorylation of Elongator by various kinases influence the recruitment of tRNA, Kti12, Hrr25/Kti14 and the phosphatase Sit4p?

How will we get to the results?

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.

Team Members

Sebastian Glatt, PhD (Project Leader)

Marcin Jaciuk, PhD (Postdoc)

Karol Zakrzewski, MSc (Scholar)

Rościsław Krutyhołowa, MSc (Scholar)

Nour El Hana Abbassi, MSc (Contractor, Scholar)

Małgorzata Honc (Contractor)

Sebastian Guzman Perez (Contractor)

Lukas Lorenz (Contractor)

How will the results influence future research on the development of science?

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.

Budget

Publications

  • Krutyhołowa R, Reinhardt-Tews A, Chramiec-Głąbik A, Breunig KD, Glatt S; Fungal Kti12 proteins display unusual linker regions and unique ATPase plops; Current Genetics 2020.
  • Pabis M, Termathe M, Ravichandran KE, Kienast SD, Krutyhołowa R, Sokołowski M, Jankowska U, Grudnik P, Leidel SA and Glatt S; Molecular basis for the bifunctional Uba4–Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation; EMBO Journal 2020.
  • Reinhardt-Tews A, Krutyhołowa R, Günzel C, Roehl C, Glatt S* and Breunig KD; A double role of the Gal80 N-terminus in activation of transcription by Gal4p; Life Science Alliance 2020.
  • Abbassi NE, Biela A, Glatt S and Lin TY; How Elongator Acetylates tRNA Bases; International Journal of Molecular Sciences 2020.
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