The Cusack group uses X-ray crystallography to study the structural biology of protein-RNA complexes involved in RNA metabolism, translation, RNA virus replication and innate immunity.
Figure: Model of the activated state of RIG-I with bound dsRNA (centre) and ATP (top-right). The helicase domains (green and cyan), the insertion domain (yellow) and the C-terminal domain (gold) all contribute to RNA binding, which displaces the CARD domains thus allowing downstream signalling and interferon expression.
Previous and current research
Aminoacyl-tRNA synthetases play an essential role in protein synthesis by charging specifically their cognate tRNA(s) with the correct amino acid. Structural information can help understand the catalytic mechanism of the enzymes and their substrate specificity for ATP, cognate amino acid and tRNA. In recent years we have focussed on leucyl-tRNA synthetase which contains a large inserted ‘editing’ domain able to recognise and hydrolyse mischarged amino acids – a proof-reading activity that is essential for maintaining translational fidelity. We have structurally characterised the large conformational changes required to switch from the aminoacylation to the editing configurations. We collaborated in the elucidation of the mechanisms of a new boroncontaining anti-fungal compound (now in Phase III clinical trials) that targets the editing site of leucyl-tRNA synthetase and helped design related anti-bacterial compounds that are active against multi-drug resistant strains, including tuberculosis.
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 cap-bound human CBC, a 90 KDa heterodimeric protein, and have studied several other proteins involved in cap-dependent processes. Once in the cytoplasm, mRNAs are subject to a quality control check to detect premature stop-codons. In all eukaryotic organisms studied, this process, known as nonsense-mediated decay (NMD), crucially depends on the three conserved Upf proteins (Upf1, Upf2 and Upf3). In mammals, it is linked to splicing. We obtained the first structural information on binary complexes of these three proteins whose ternary complex formation triggers decay.
Future projects and goals
Ongoing projects related to RNA metabolism include continued studies on PHAX and ARS2, both of which bind CBC and are linked to the metabolism of small RNAs. A major focus is structure determination of the influenza virus RNA-dependent RNA 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 permit structure-based antiviral drug design. To pursue this we have co-founded a Vienna-based company called SAVIRA. 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 engaged in confocal and cross-correlation fluorescence studies as well as correlative EM microscopy of the assembly and trafficking of the influenza polymerase and RNPs in living, infected cells. Another major project is innate immune pattern recognition receptors such as NOD proteins and RIG-I like helicases. In 2011 we published the first structure-based mechanism of activation of RIG-I and are continuing to study this signalling pathway. Finally we have new projects on epigenetic complexes involving the histone acetylase MOF, such as the X-chromosome dosage compensation or male-specific lethal complex, which in Drosophila contains an essential non-coding RNA and the more recently discovered NLS complex (with the Akhtar group, MPI, Freiburg).