Figure 1: Structure of the 44-subunit catalytic step 1 spliceosome determined by cryo-EM to 3.8 Å resolution.

Figure 1: Structure of the 44-subunit catalytic step 1 spliceosome determined by cryo-EM to 3.8 Å resolution.

Figure 2: A network of RNA-RNA interactions between pre-mRNA substrate and snRNAs at the spliceosomal active site.

Figure 2: A network of RNA-RNA interactions between pre-mRNA substrate and snRNAs at the spliceosomal active site.

The Galej group uses an integrated structural biology approach combined with biochemistry and biophysics to investigate large RNA-protein complexes involved in gene expression.

Previous and current research

The spliceosome is a large and dynamic RNA-protein complex, which catalyses excision of non-coding regions (introns) from the precursors of messenger RNAs (pre-mRNAs). During my PhD, I investigated the structure and function of the largest and the most highly conserved spliceosomal protein – Prp8. I crystallised and solved the structure of a 220-kDa Prp8:Aar2 complex at 1.9 Å resolution (Galej et al., 2013). This structure provided the first direct insights into the organization of the protein component of the spliceosomal active site and revealed unexpected evolutionary links to self-splicing group II intron encoded proteins (IEP).

In the past few years, a technological revolution in the field of cryo-EM allowed us to look into much more complex and dynamic molecular assemblies. We determined the first medium-resolution (5.9 Å) cryo-EM structure of the spliceosomal component – U4/U6.U5 tri-snRNP, followed by a complete, near-atomic resolution structure of this 1.5 MDa RNA-protein complex (Nguyen et al., 2016). The structure of the tri-snRNP provided unprecedented insights into the molecular architecture of the spliceosome at the pre-catalytic stage of its assembly.

Finally, we established a protocol for a large scale purification of entire spliceosomes assembled in vitro in a whole cell extract. We trapped spliceosomes immediately after the first catalytic reaction and solved the structure by cryo-EM to 3.8 Å resolution (Galej et al., 2016). Our structure of the 44-subunit catalytic, step 1 spliceosome revealed organization of the active site and the role of step 1 protein factors in juxtaposition of the splice sites. The presence of a DEAH-box helicase in close proximity to the active site suggests a possible mechanism of remodelling between the two catalytic steps of splicing.

My group is investigating the mechanism underlying complex hierarchical assembly of snRNPs and spliceosomes. In our laboratory, we use an integrative structural biology approach with the main focus on cryo-EM studies of large RNA-protein complexes. We use X-ray crystallography to determine high-resolution structures of small proteins and single domains. Structural studies are complemented with biochemical and biophysical experiments, including Electrophoretic Mobility Shift Assays (EMSA), Isothermal Titration Calorimetry (ITC), Cross-linking and Mass Spectrometry (MS-XL). Integral parts of our research are functional studies, which include an in vitro depletion/reconstitution approach and in vivo genetics. We are also planning to employ single molecule technologies and next generation RNA sequencing methods to broaden our understanding of the dynamics of the investigated systems.

Future projects and goals

  • Cryo-EM and crystallographic studies of complexes involved in snRNA processing and biogenesis of spliceosomal snRNPs.
  • In vitro reconstitution of snRNPs assembly pathways.
  • Structural studies of the spliceosome assembly intermediates.