Figure 1: Crystal structure of the 10-subunit yeast exosome bound to a T4-lysozyme fused Ski7 (turquoise) and RNA (black). Core subunits of the exosome are grey, cap subunits in shades of yellow and the nuclease Rrp44 is shown in pink.
The Kowalinski group investigates the architecture and mechanisms of macromolecular complexes involved in cellular RNA editing and modification.
Previous and current research
Post-transcriptional chemical RNA modifications are ubiquitous (tRNA, mRNA, rRNA, snoRNA, etc.) and occur in all kingdoms of live. For example, eukaryotic tRNAs contain on average 13 modifications per molecule that assure translation fidelity and efficiency. Modifications on mRNAs have effects on folding, solubility, transcript stability, splicing and RNA localization. Thus, the so called “epitranscriptome” adds an additional layer of information to the message. This layer is reversible in response to cellular stress, metabolic changes or developmental stage. In the Kowalinski lab we are investigating macromolecular complexes that modify RNA or recognize modified RNAs. We solve structures of these complexes by single particle cryo electron microscopy (cryo-EM) and x-ray crystallography to understand the following questions: How exactly is a specific RNA is selected for modification? How is this process is regulated? How does a modified RNA differ in its binding to downstream effectors?
During my PhD thesis I investigated how the innate immune receptor RIG-I triggers an inflammatory response upon recognition of viral RNA. Solving several crystal structures of RIG-I with and without ligand RNA enabled us to draw a conclusive picture of the conformational changes that lead to activation of the receptor (Kowalinski et al., Cell, 2011). In my postdoctoral work, I studied how the helicase-containing Ski complex aids the degradation of cellular RNAs by the exosome complex. Based on the structure of the exosome bound to Ski7 we understood how compartment specificity of the exosomal co-factors is achieved and moreover could identify the human homologue on evidence of the structure (Kowalinski et al., MolCell, 2016). In collaborative efforts we found that the cellular mRNA degradation machinery (the exosome via the Ski-complex) directly interacts with the cellular translation machinery (the ribosome) (Schmidt, Kowalinski et al., Science 2016).
Future projects and goals
In the future, the Kowalinski group wants to gain insight into the mechanisms that specify certain RNAs for modification. We would like to understand how RNA editing and modification reactions are controlled and influenced by other factors and how a modified RNA behaves differently. To this end, we will not only reconstitute recombinant complexes in vitro but also purify native endogenous complexes for structural investigation. We will use X-ray crystallography, cryo-electron microscopy and scattering techniques like SAXS or SANS combined with biophysical methods and biochemical assays to assess the structure-function relationship within these complexes. We will study Trypanosoma brucei, the causative agent of the sleeping sickness, as a model system. Later on, we would like to also study dynamic details with single molecule techniques as well as systemic effects via RNA sequencing techniques.