Cusack Group
Structural biology of RNA-protein complexes in gene expression and host-pathogen interactions
Ribbon diagram showing the cap-binding domain of influenza virus polymerase subunit PB2 (yellow and red) with bound cap analogue (purple).
Previous and current research
We use X-ray crystallography as a central technique to study the structural biology of protein-RNA complexes involved in RNA metabolism, translation, virus replication and epigenetics. Current major focuses are on negative strand RNA virus polymerases, innate immune system receptors and the dosage compensation complex.
The nuclear cap-binding complex (CBC) binds to m7Gppp cap at the 5’ end of Pol II transcripts and mediates interaction with nuclear RNA processing machineries for splicing, poly-adenylation and transport. We determined the structure of human CBC, a 90KDa heterodimeric protein and its complex with a cap analogue and are currently working on structures of several other proteins involved in cap-dependent processes. We have also worked on the structure of the protein PHAX, which binds to CBC and is specifically involved in nuclear transport and export of small capped non-coding RNAs (e.g. snRNAs). Once in the cytoplasm, mRNAs are subject to a quality control check to detect premature stop-codons. This process, known as nonsense mediated decay (NMD), crucially depends on the three proteins Upf1, Upf2 and Upf3 in all eukaryotic organisms studied, and in mammals is linked to splicing. We obtained the first structural information on the interacting domains of these three proteins whose ternary complex formation triggers decay.
Aminoacyl-tRNA synthetases play an essential role in protein synthesis by charging specifically their cognate tRNA(s) with the correct amino acid. We aim to obtain structural information to help understand the catalytic mechanism of the enzymes and their substrate specificity for ATP, cognate amino acid and tRNA. Most recently we solved the structures of a class I enzyme, leucyl-tRNA synthetase, and a class II enzyme prolyltRNA synthetase, each with their cognate tRNAs bound. Both these enzymes contain a large inserted editing domain able to recognise and hydrolyse mischarged amino acids. This proof-reading activity is essential for maintaining translational fidelity. We have collaborated in the elucidation of the mechnisms of a new antifungal compound that targets the editing site of leucyl-tRNA synthetase and have now extended this using structure-based approaches to design new anti-bacterials that are active against multi-drug resistant strains.
Future projects and goals
We have several ongoing projects related to RNA metabolism, aiming to obtain and use structures of the complexes involved to understand function. These include continued studies on PHAX and ARS2, both of which bind CBC and are linked to the metabolism of small RNAs. Concerning NMD, we published the structure of the UPF1-UPF2 complex and are now determining the architecture of the entire trimeric UPF1-UPF2-UPF3 complex. A major ongoing project is structure determination of the trimeric influenza virus RNA-dependent polymerase, the viral replication machine.
We have determined the structure of four distinct domains from the polymerase, including the two key domains involved in the ‘cap-snatching’ process of viral mRNA transcription: the cap-binding site in PB2 and the endonuclease in PA. These results give some insight into the polymerase mutations required to adapt an avian virus to be able infect humans and also give a boost to structure based antiviral drug design (we co-founded a company to pursue this). This work is now being extended to the polymerases of other segmented RNA viruses such as bunyaviruses which also perform cap-snatching. In collaboration with the Ellenberg and Briggs groups we are now engaged in confocal and cross-correlation fluorescence studies as well as correlative EM microscopy of the transport and assembly of the influenza polymerase and RNPs in living, infected cells.
We also work on the structure and mechanism of activation of Rig-I, an intracellular pattern recognition receptor of the innate immune system which signals interferon production upon detection of viral RNA. Finally we have two new projects concerning ncRNAs: the structure and mechanism of the X-chromosome dosage compensation (MSL complex), which in Drosophila contains an essential non-coding RNA and secondly, in collaboration with the Pillai group (page 98), structural studies of proteins involved in the piRNA pathway.