About us

 

We study different translation control mechanisms, which regulate the production of specific sets of proteins by chemical modifications of tRNA molecules. Every protein in the cell is produced by the ribosome, which uses transfer RNA (tRNA) molecules to translate the sequence information coded in mRNAs into correctly assembled poly-peptide chains. The decoding/translation of genetic information is based on the recognition of a respective codon by its corresponding tRNA anticodon triplet.

The lab is focusing on understanding the molecular mechanisms that lead to the specific base modifications in anticodons of tRNAs. These modifications have a strong influence on the efficiency and accuracy of the codon-anticodon pairing and therefore regulate the translational rates and folding dynamics of protein synthesis. Recent findings have shown that alterations of these modification pathways play important roles in the onset of certain neurodegenerative diseases and cancer.

crystalographycrystalscrystal

   Protein crystallography enables us to visualize protein structures at the atomic level and enhances our understanding of protein function. X-ray crystallography was first applied for biological macromolecules (i.e. hemoglobin) in the late 1950s and had been awarded Nobel Prize in Chemistry in 1962, which had a great impact on further development of structural biology. In order to determine the structure, a high-quality crystal of the specific protein is required. Obtaining such crystals is a crucial step in protein crystallography and has been fully automated by developing crystallization platforms, which perform high-throughput screening of hundreds of thousands of experimental conditions to facilitate the optimization of crystallization process. To see proteins in atomic resolution and determine their structure, we need to use electromagnetic radiation provided by synchrotron beamlines. This advanced technique is based on X-rays scattering on molecule atoms that provide a diffraction pattern, used to generate an electron density map and eventually solve the protein structure.

   In our laboratory, we use X-ray crystallography to study how proteins interact with other molecules (i.e. tRNA) and gain detailed knowledge about macromolecular assemblies and their role in post-transcriptional regulation. Recently, we have obtained the TEAM-TECH Core Facility grant from Foundation for Polish Science to establish a Structural Biology Core Facility, equipped with highly specialized devices for crystallization trials to reassure maximum efficiency and reproducibility in crystallization pipeline.

tRNA modifications

   tRNA modification is a complex field regarding the fact that over 100 different kinds of modifications have been discovered and their diverse roles in maintaining tRNA structure rigidity and ensuring the translation fidelity during decoding in ribosome. We are interested in the Elongator complex-mediated cm5 modification on the uridine 34 at wobble position of tRNA anticodon loop. The lack of this modification disables further modifications, such as mcm5, ncm5, mcm5s2, and this has been correlated to pleiotropic phenotypes in cellular level as well as in human diseases, including cancers, neurodegenerative diseases and intellectual disabilities. The Elongator complex is highly conserved and it is consist of 2 copies of 6 subunits (Elp1-6). The integrity of the complex is tightly connected to its function. We recently reported the EM structure of the apo complex as well as the crystal structure of the catalytic subunit (Elp3). With biophysical and biochemical analyses, we could explain how this catalytic subunit interacts with its tRNA substrate as well as how other subunits contribute to its proper function.

   For more information, please check out these articles and reviews!

Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi

tRNA Modification by Elongator Protein 3 (Elp3)

Structural asymmetry in the eukaryotic Elongator complex

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior

gridelongator-structureelectron-microscopy

 Single particle cryo-electron microscopy (cryo-EM) is a structural biology method used for solving high resolution structures of proteins and their complexes. Due to recent advances in hardware and software it becomes more and more popular. In comparison to crystallography, where crystal growth constitutes a serious bottleneck of structure solution process, in cryo-EM protein particles are frozen in liquid like state of vitreous ice and imaged under electron microscope. Following that, particles are selected and classified to generate 2d classes. Next these 2d classes are used to reconstitute density maps of the studied particles which then can be further used for model building.

   In Max Planck Research Group we are utilising cryo-EM to study 850 kDa eukaryotic Elongator complex responsible for initial modification of wobble uridine at C5 position of selected tRNAs. In addition to that we are also interested in how Elongator is regulated by Kti11, Kti12, Kti13 and Kti14 proteins. Structural biology core facility in Malopolska Center of Biotechnology of Jagiellonian University (JU) is equipped with JEOL HDT-400 glow discharger, FEI Vitrobot Mark IV and Molecular Dimensions grid box storage system, everything that is needed to prepare and store cryo-EM samples. Cryo grids are screened on JEOL JEM2100 HT CRYO LaB6 electron microscope installed in the Department of Cell Biology and Imaging in the Institute of Zoology and Biomedical Research JU.

   Cell Room Team was established as a part of a First Team Project to investigate the role of Elongator complex in human cells. We work mainly on HEK293 cells that are our source of human tRNAs and human proteins involved in tRNA modification process. Our main techniques are transfection and lentiviral transduction to modulate levels of proteins of interest; WB, RT-PCR, fluorescent confocal microscopy to verify protein levels and their localisation. Apart from basic phenotype tests (cell viability, cell proliferation), we have also implemented HPLC analysis and specific tests to quantify levels of modified tRNA isoacceptors and multiple functional tests to monitor consequences of loss of Elongator and/or its cofactors function (response to oxidative stress, protein aggregation, migration). Finally, we are interested in interactions between human proteins involved in tRNA modification process (CoIP, BioID2 on wild type or mutant proteins overexpressed in cells).

Grants

  • EMBO Installation Grant 2015 – 2018
  • NCN OPUS10 NZ 2016 – 2019
  • NCN POLONEZ1 (Monika Gaik) 2016 – 2018
  • FNP FirstTEAM 2016 – 2019
  • FNP Homing (Ting-Yu Lin) 2017 – 2019
  • TEAM TECH Core Facility grant 2018 – 2021 

Publications

  1. Kojic M, Gaik M, Kiska B, Salerno-Kochan A, Hunt S, Tedoldi A, Mureev S, Jones A, Whittle B, Genovesi LA, Adolphe C, Brown DL, Stow JL, Alexandrov K, Sah P, Glatt S*, Wainwright BJ*; Elongator mutation in mice induces neurodegeneration and ataxia-like behavior; Nat Comm. 2018 Aug;9(1)
  2. Lin TY and Glatt S, tRNA modification by Elongator protein 3 (Elp3), Encyclopedia of Inorganic and Bioinorganic Chemistry 2018 (book chapter)
  3. Sokołowski M*, Klassen R*, Bruch A, Schaffrath R, Glatt S; Cooperativity between different tRNA modifications and their modification pathways; Biochim Biophys Acta. 2018 Apr;1861(4)
  4. Dauden MI*, Jaciuk M*, Müller CW, Glatt S; Structural asymmetry in the eukaryotic Elongator complex, FEBS Lett. 2017 Sep;592(4)
  5. Mehlgarten C, Prochaska H, Hammermeister A, Abdel-Fattah W, Wagner M, Krutyhołow R, Jun SE, Kim GT, Glatt S, Breunig KD, Stark MJR, Schaffrath R; Use of a yeast tRNase killer toxin to diagnose Kti12 2 motifs required for tRNA modification by Elongator, Toxins 2017 Sep;9(9).
  6. Dauden MI, Kosinski J, Kolaj-Robin O, Desfosses A, Ori A, Faux C, Hoffmann NA, Onuma OF, Breunig KD, Beck M, Sachse C, Seraphin B, Glatt S*, Müller CW*; Architecture of the yeast Elongator complex, EMBO Rep. 2017 Feb;18(2)
  7. Glatt S, Zabel R, Kolaj-Robin O, Onuma OF, Baudin F, Graziadei A, Taverniti V, Lin TY, Baymann F, Séraphin B, Breunig KD, Müller CW; Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi. Nat Struct Mol Biol. 2016 Sep;23(9)
  8. Luna-Zurita L, Stirnimann CU, Glatt S, Kaynak BL, Thomas S, Baudin F, He D, Small EM, Mileikovsky M, Holloway AK, Nagy A, Pollard KS, Müller CW & Bruneau BG; Cooperative interactions and locus tethering as the basis for regulation of cardiogenesis by heterotypic transcription factors; Cell 2016 Feb 25; 164 (5)
  9. Silva-Martin N*, Daudén MI*, Glatt S, Hoffmann NA, Kastritis P, Bork P, Beck M and Müller CW.; The combination of X-ray crystallography and cryo-electron microscopy provides insight into the overall architecture of the dodecameric Rvb1/Rvb2 complex; PLoS One 2016 Jan 8; 11 (1)
  10. Hoffmann NA, Jakobi AJ, Moreno-Morcillo M, Glatt S, Kosinski J, Hagen WJH, Sachse C, Müller CW; Molecular structures of unbound and transcribing RNA polymerase III; Nature 2015 Dec 10; 528 (7581)
  11. Jeske M, Bordi M, Glatt S, Müller S, Rybin V, Müller CW and Ephrussi A; The crystal structure of the Drosophila germ line-inducer Oskar identifies two domains with distinct Vasa helicase- and RNAbinding activities; Cell Rep. 2015 Jul 28; 12(4)
  12. Male G, von Appen A, Glatt S, Taylor NMI, Cristovao M, Groetsch H, Beck M and Müller CW; Architecture of TFIIIC and the role of τ131 in RNA polymerase III pre-initiation complex assembly; Nat Comm. 2015 June 10; 6
  13. Glatt S*, Zabel R*, Vonkova I, Kumar A, Netz D, Pierik A, Lill R, Gavin AC, Balbach J, Breunig KD, Müller CW; Kti11/Kti13 structure reveals its dual its role in Elongator dependent tRNA modification  and diphthamide biosynthesis; Structure 2015 Jan 6; 23(1)
  14. Singh B, Glatt S, Ferrer JL, Dunder J, and Hansman GS; Structural implications of feline norovirus protruding domain; Virology 2015 Jan 1; 474
  15. Alfieri C, Gambetta MC, Matos R, Glatt S, Sehr P, Fraterman S, Wilm M, Müller J. & Müller CW; Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements; Genes Dev. 2013 Nov 1;27(21)
  16. Taylor NM, Glatt S, Hennrich ML, von Scheven G, Grotsch H, Fernandez-Tornero C, Rybin V, Gavin AC, Kolb P & Müller CW; Structural and functional characterization of a phosphatase domain within yeast general transcription factor IIIC; J Biol Chem. 2013 May 24;288(21)
  17. Glatt S, Müller CW; Structural insights into Elongator function; Curr Opin Struct Biol. 2013 Apr;23(2)
  18. Glatt S, Séraphin B, Müller CW; Elongator: transcriptional or translational regulator? Transcription. 2012 Aug 14;3(6)
  19. Glatt S, Létoquart J, Faux C, Taylor N, Séraphin B and Müller CW; The Elongator subcomplex Elp456 is a hexameric RecA-like ATPase; Nat Struct Mol Biol. 2012 Feb 19;19(3);
  20. Glatt S, Alfieri C, Müller CW; Recognizing and remodeling the nucleosome; Curr Opin Struct Biol. 2011 Jun; 21(3)
  21. Glatt S, Halbauer D, Heindl S, Wernitznig A, Kozina D, Su KC, Puri C, Garin-Chesa P, Sommergruber W; hGPR87 contributes to viability of human tumor cells; Int J Cancer. 2008 May 1; 122(9);
  22. Dohnal AM, Inthal A, Felzmann T, Glatt S, Sommergruber W, Mann G, Gadner H and PanzerGrümayer ER; Leukemia-associated antigenic isoforms induce a specific immune response in children with T-ALL; Int. J. Cancer. 2006: Dec 15;119(12)