Structural biology; nucleic acid regulation; RNAi; molecular recognition; X-ray crystallography
We study the molecular basis of nucleic acid regulatory processes, RNAi and DNA replication in particular. We use the tools of structural biology, biochemistry and biophysics to study proteins and protein complexes associated with these processes to elucidate how they work. X-ray crystallography, EM, and other structural techniques enables us to obtain the three-dimensional structures of these molecular machines. Biochemistry, biophysics and molecular biology allow us to study properties that can be correlated to their function and biology.
The introduction of exogenous double-stranded RNA (dsRNA) into a cell can trigger the gene silencing process called RNA interference or RNAi. Although there has been remarkable progress in unraveling the components of the RNAi machinery, in order to get a complete understanding of these mechanisms we must determine how they work at the molecular level. Therefore, we embarked on structural and biochemical studies of these proteins. By solving the structure of a full-length Argonaute protein, the key component in the RNAi machinery, we identified Argonaute as “Slicer”, the effector enzyme that harbors the small RNAs, e.g. miRNAs and siRNAs, and cleaves the mRNA as directed by the siRNA. These studies enhance not only our understanding of this important pathway, but should also improve the practical use of the RNAi technology as an experimental tool for gene knockdown technology.
Another area of research in the lab is DNA replication. The goal is to understand the molecular motors involved in replication initiation. We have been studying the viral replicative helicase E1 from papillomavirus as a model system to study helicase function and assembly as well as DNA melting and unwinding. We have also been examining the eukaryotic machinery with the Origin Recognition Complex (ORC) as the centerpiece of these studies.
Kuhn, C. D. and Wilusz, J. E. and Zheng, Y. and Beal, P. A. and Joshua-Tor, L. (2015) On-Enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-Adding Enzyme. Cell, 160 (4). pp. 644-658. ISSN 0092-8674
Faehnle, C. R. and Walleshauser, J. and Joshua-Tor, L. (2014) Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway. Nature, 514 (7521). pp. 252-6. ISSN 0028-0836
Lee, S. J. and Syed, S. and Enemark, E. J. and Schuck, S. and Stenlund, A. and Ha, T. and Joshua-Tor, L. (2014) Dynamic look at DNA unwinding by a replicative helicase. Proceedings of the National Academy of Sciences of the United States of America, 111 (9). E827-35. ISSN 0027-8424
Kuscu, C. and Zaratiegui, M. and Kim, H. S. and Wah, D. A. and Martienssen, R. A. and Schalch, T. and Joshua-Tor, L. (2014) CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing. Proc Natl Acad Sci U S A. ISSN 0027-8424
C. Shen, J. J. Ipsaro, J. Shi, J. A. Milazzo, E. Wang, J.-S. Roe, Y. Suzuki, D. J. Pappin, L. Joshua-Tor, and C. R. Vakoc, NSD3-short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler, Mol. Cell, 60, published online November 25, 2015.