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Professor Christiane Berger-Schaffitzel

Professor Christiane Berger-Schaffitzel

Professor Christiane Berger-Schaffitzel
MSc(Hannover), PhD(Zurich), Habiliation (E.T.H.Zurich)

Professor of Biochemistry

Area of research

Translation, Protein and mRNA Quality Control

Office B floor
Biomedical Sciences Building,
University Walk, Clifton BS8 1TD
(See a map)

+44 (0) 117 39 41869

Summary

Current work in the lab ranges from eukaryotic translation initiation, via regulation of translation termination to co-translational translocation of membrane proteins via the bacterial holo-translocon. We are particularly interested in how cells recognize problems during protein synthesis and target the defective proteins and their encoding mRNAs to degradation. These processes are vital to all organisms, and even minor problems in mRNA or protein quality control mechanisms give rise to diseases. Our research is funded by the BBSRC and MRC and recently via a Wellcome Trust Investigator Award.

In all our projects, we rely on biochemical methods, using in vitro translation systems. Most projects in the lab also involve biophysics and structural biology, crystallography and cryo-EM. We have established a Wellcome Trust-funded GW4 Facility for high-resolution Cryo-EM with a 200kV Talos Arctica with energy filter and K2 Direct Electron Detector. Image processing is supported by a BBSRC-funded BlueCryo high-performance computing cluster. 

Biography

Projects

mRNA Quality Control Mechanisms

Nonsense-mediated mRNA decay (NMD) is an essential mechanism controlling translation in the eukaryotic cell. NMD ascertains accurate expression of the genetic information by quality controlling messenger RNA (mRNA). During translation, NMD factors recognize and target to degradation aberrant mRNAs that have a premature stop codon (PTC) and that would otherwise lead to the production of truncated proteins which could be harmful for the cell. Discrimination of a PTC from a correct termination codon depends on splicing and translation, and it is the first and foremost step in human NMD. However, the underlying molecular mechanisms remain elusive as we do not know how the NMD factors orchestrate the vital step of recognising faulty mRNAs.

Recently, we discovered novel NMD factor interactions and new functions of a known NMD factor, which may unlock this enigma. We now test these interactions in living cells and in vitro. We use crystallography and electron cryo-microscopy to visualise how these proteins function and thus reveal the interplay of the protein-synthesis and the quality-control machinery, in health and disease.

 

 Figuere1

Caption: We study the interplay of the translation machinery (terminating ribosome) and NMD factors to understand how a faulty mRNA is recognised using biochemistry – e.g. primer extension assays shown on the left - and cryo-EM.

Further Reading:

Phosphatidylinositol-3-kinase related-kinases

SMG-1 kinase is essential for human NMD and member of the phosphatidylinositol-3-kinase related-kinase family (PIKKs). Phosphorylation of NMD factor UPF1 by SMG1 is suggested to trigger NMD. We have solved a low-resolution cryo-EM structure of the SMG1-8-9-UPF1 complex and found that SMG1's C-insertion domain regulates kinase activity (NAR 2015). We further solved the structure of the conserved UPF2 MIF4G domains and described UPF2’s impact on SMG1 kinase activity (NAR 2013,2015). 

Like SMG1, Target of Rapamycin is a PIKK. It exists in two complexes: TORC1 and TORC2. We solved the cryo-EM structure of yeast TORC2, which suggests how substrates are recruited to the kinase and revealed the molecular basis of rapamycin resistance of TORC2 (Mol Cell 2015, Nat Commun. 2017).

 Figuere2

Caption: Structural characterisation of PIK-like kinases. a, NMD factor complex SMG-1, SMG-8, SMG-9 and UPF1 was purified, reconstituted and characterised by cryo-EM and crosslinking mass spectrometry. b, 7.9 Å-resolution cryo-EM structure of Saccharomyces cerevisiae TOR Complex 2 providing first insights into the molecular basis of rapamycin insensitivity of this 1.4 MDa complex (below).

Further Reading:

The bacterial translocation machinery

Targeting and translocation systems are required to transport proteins into the membrane or across the membrane to the cellular location where they can fulfil their tasks. These systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed. The Sec translocation system is well studied and conserved from the bacteria to humans highlighting its importance.

Current research on protein translocation focused on the structure and function of the conserved heterotrimeric protein translocation pore – SecYEG in E. coli. Little is known about the structure and function of the additional components of the bacterial translocation machinery, SecD, SecF and YidC, which are essential for E. coli survival. In spite of their central role, much less is known about how these proteins work.

This is to a large part due to the lack of structural data of larger transmembrane complexes comprising SecYEG and these subunits. Using a new recombineering-based vector system for expression of multi-protein complexes in E. coli, we over-produced a super-complex of this SecYEG-DF-YidC holo-translocon (HTL). We also succeeded in detergent-solubilising and purifying this complex. With this purified holo-translocon, we pursue a thorough functional and structural characterization of this vitally important membrane protein complex. The central aim is to determine high-resolution structures of the holo-translocon alone and in physiologically important complexes by cryo-EM. We further aim to understand how proteins that cannot correctly fold are recognized by the cellular quality control system. We will elucidate how these translocation and quality control machines work together in the membrane to ensure the proper folding of membrane proteins and to remove unfolded proteins that could be detrimental to the cell.

 

 Figuere3

Caption: a, Schematic representation of the pACEMBL_HTL expression plasmid (for details see Bieniossek et al., 2009). b, left: Coomassie-stained SDS-PAGE of the holo-translocon (HTL). Right: Western blot detection of the CBP-tagged YajC protein. c, low resolution cryo-EM reconstruction of E. coli HTL displayed in different views and placement of high-resolution structures of SecYEG (blue) and SecDF (green) based on crosslinking and EM localization experiments (Botte et al., 2016). 

Further Reading:

Keywords

  • Nonsense-Mediated messenger RNA Decay
  • Eukaryotic Translation Initiation and Termination
  • Human Ribosome
  • Membrane Protein Synthesis
  • Folding and Quality Control
  • In vitro Translation
  • Cryo-EM and Image Processing

Memberships

Organisations

School of Biochemistry

Other sites

School of Biochemistry staff

Selected publications

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View complete publications list in the University of Bristol publications system

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