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Clemens Plaschka

Messenger RNA (mRNA) maturation is a crucial step in gene expression, requiring the coordinated action of several macromolecular complexes. The most dramatic maturation event is the removal of introns from pre-mRNA by the spliceosome, a large and dynamic ribonucleoprotein enzyme. We apply structural and functional methods to elucidate the molecular basis of spliceosome regulation and its coordination with other maturation events. Our goal is to obtain snapshots of the different stages of mRNA maturation to understand how a functional mRNA is produced.

Eukaryotic mRNA expression begins with gene transcription in the nucleus. The nascent precursor mRNA (pre-mRNA) undergoes co- and post-transcriptional processing, which is critical for producing the mature mRNA. The mRNA can then be exported and translated in the cytoplasm. Over the course of maturation the mRNA is never left exposed, but is instead bound by protein factors and complexes that exchange dynamically. This facilitates the maturation itself as well as coordination between the different maturation steps. Our lab aims to understand in molecular detail how nuclear macromolecular complexes facilitate mRNA processing and thereby regulate gene expression.

Figure 1: Single particle cryo-EM analysis of RNA-protein complexes, an example from the spliceosome. To study spliceosome assembly, splicing intermediates are stalled biochemically and purified to homogeneity (left). The purified complexes are flash-cooled in a thin layer of vitreous ice, imaged using a cryo-electron microscope, and the resulting data processed (left of centre). Many thousand single particle images are then refined to reconstruct a cryo-EM density of the spliceosome to high resolution, enabling its structural modelling (right of centre). The final structure visualizes a snapshot of the spliceosome in action, revealing new insights on its mechanism and its regulation (right). Adapted from Plaschka et al., Nature (2017) 546, 617-621 and Wilkinson et al., Annu Rev of Biophys (2018) in press.

A key mRNA maturation event is the splicing of the pre-mRNA. Splicing is carried out by the spliceosome, a dynamic multi-megadalton machine that excises non-coding introns from the pre-mRNA. The splicing machinery assembles anew on each intron through the step-wise addition of five U-rich small nuclear RNA–protein complexes (U1, U2, U4, U5, U6), and several accessory factors, comprising over 70 proteins in yeast and over 100 proteins in humans. During spliceosome assembly, alternative exons can be selected, leading to distinct mRNA isoforms from a single gene. These mRNA isoforms can encode for proteins with dramatically different functions. Splicing additionally coordinates with downstream events, such as the packaging and export of mRNA. Despite the important roles of these maturation mechanisms in gene expression, many questions regarding their molecular details remain. What is the structural and mechanistic basis of splicing regulation? How do splicing factors establish alternative splicing programs? How are pre-mRNA splicing and downstream events coordinated? How are defects in the mRNA life cycle linked to human disease?

 
Figure 2: Cryo-EM structure of the spliceosome B complex. The structure contains the pre-precursor mRNA substrate (pre-mRNA, black), 52 spliceosome proteins, and four small nuclear RNAs that are coloured according to ribonucleoprotein complex identity (U2, green; U4, yellow; U5, blue; U6, red). For details see Plaschka et al., Nature (2017) 546, 617-621.

To shed light on these questions, we determine the three-dimensional structures of yeast and human mRNA maturation intermediates. We prepare mRNA-protein complexes from endogenous or recombinant sources and use single particle cryo-electron microscopy (cryo-EM) and X-ray crystallography resolve their structures. Complementary methods, such as protein crosslinking and mass spectrometry, facilitate an integrative modelling of the complexes. By combining these structural studies with functional biochemistry, we further elucidate the molecular mechanisms in vitro. Through these approaches we have previously revealed mechanisms of RNA polymerase II transcription (Lariviere*, Plaschka*, et al., Nature 2012; Plaschka et al., Nature 2015; Plaschka*, Hantsche* et. al, Nature 2016; see below) and of pre-mRNA splicing (Plaschka*, Lin* et al., Nature 2017; Plaschka*, Lin* et al., Nature 2018; see below).

We welcome applications from pre- and post-doctoral scientists, who wish to apply state-of-the-art structural biology methods to understand the dynamic and complex processes of mRNA regulation.

Moreover, we are advertising for a Master student position, which is available until filled.

Selected Publications

  • C. Plaschka*#, P.-C. Lin*#, C. Charenton, K. Nagai#. Prespliceosome structure provides insights into spliceosome assembly and regulation. Nature (2018) in press. *Authors with equal contribution. #Co-corresponding authors.
  • C. Plaschka*#, P.-C. Lin*#, K. Nagai#. Structure of a pre-catalytic spliceosome. Nature (2017) 546, 617-621. *Authors with equal contribution. #Co-corresponding authors.
  • C. Plaschka*, M. Hantsche*, C. Dienemann, C. Burzinski, J. Plitzko, P. Cramer. Transcription initiation complex structures elucidate promoter DNA opening. Nature (2016) 533, 353-358. *Authors with equal contribution. 
  • C. Plaschka, L. Larivière, L. Wenzeck, M. Seizl, M. Hemann, D. Tegunov, E. V. Petrotchenko, C. H. Borchers, W. Baumeister, F. Herzog, E. Villa, P. Cramer. Architecture of the RNA polymerase II–Mediator core initiation complex. Nature (2015) 518, 376-380