Cusack Group

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 and translation. A current major focus is on influenza virus polymerase and innate immune system receptors.

In eukaryotic cells, nascent Pol II RNA transcripts (e.g. mRNA or snRNA) are rapidly given an m7Gppp cap at the 5’ end. The nuclear cap-binding complex (CBC) binds to this tag 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 have obtained the first structural information on the interacting domains of these three proteins whose ternary complex formation triggers decay.We have also determined the structure of the complete, heterodimeric, tri-functional vaccinia virus mRNA capping enzyme, which caps viral transcripts in the cytoplasm of infected cells.

Aminoacyl-tRNA synthetases play an essential role in protein synthesis by charging specifically their cognate tRNA(s) with the correct amino acid.We aimto obtain atomic resolution structural information to help us understand the catalytic mechanism of the enzymes and their substrate specificity for ATP, cognate amino acid and tRNA. Recently we have solved the structures of a class I enzyme, leucyl-tRNA synthetase, and a class II enzyme, prolyl-tRNA synthetase, each with their cognate tRNAs bound. Both contain a large inserted editing domain able to recognise and hydrolysemis charged amino acids, essential for maintaining translational fidelity.

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 recently published the structure of the UPF1-UPF2 complex, and are now trying to determine the architecture of the entire trimeric UPF1-UPF2-UPF3 complex.Work is continuing on the leucyl-tRNA synthetase systems, both of which have editing activities. We collaborated in the elucidation of the mechanism of action of a new anti-fungal compound that targets the editing site of leucyl-tRNA synthetase and are now extending this by using structure based approaches to design new anti-bacterials that are active against multi-drug resistant strains.

A major goal is the structure determination is structure determination of the trimeric influenza virus RNA-dependent polymerase, the viral replicationmachinery.We have collaborated in the structure determination of four distinct domains from the polymerase, the C-terminal domain of the PB2 subunit involved in nuclear import, the 627-domain of PB2 (which contains important host determinant, residue 627) and domains containing the two key active sites involved in the ‘capsnatching’ 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). We are also engaged in fluorescence studies of the transport and assembly of the influenza polymerase in living cells. Finally we work on the structure and mechanismof activation of intracellular pattern recognition receptors of the innate immune system such as the NOD proteins, which respond to fragments of bacterial cell walls and the RIG-I like helicases, which signal interferon production upon detection of viral RNA.