My CV can be found here.
I have studied the structural basis of RNA function, protein-RNA and RNA-small molecule recognition since 1987, first as a post-doctoral fellow in Berkeley (1987-1992), then as a Faculty at the MRC in Cambridge (1992-2001) and in Seattle (2001-). I have studied RNA structures and RNA-protein complexes involved in gene expression in eukaryotes and RNA viruses, and have applied this understanding to investigate the molecular basis of human disease and to seek new therapeutic avenues to treat infectious diseases. Among my accomplishments are the structures of the two most common building blocks of RNA secondary structure (the super stable UUCG tetraloop (1) and the E- or S-loop (2)); two of the very first structures of the two largest families of RNA-binding proteins (RRM (3) and dsRBD (4)); very early studies of RNA and protein dynamics (5). These studies required the development and implementation of new spectroscopic NMR methods as well, which we accomplished, in some cases, years ahead of other researchers (6).
Among my continuing interests is exploiting the structural knowledge we generate to treat human disease (7-10). I have done so in an Academic context and as consultant and founder of a successful biotechnology start-up, which became a public company. Our most recent results in this area are particularly promising: we have discovered inhibitors of protein-RNA interactions with low picomolar binding activity, unprecedented specificity and cellular potency comparable to FDA-approved antivirals (11,12), a set of goals I have pursued since 1991. Herein I use a rather broad brush to briefly summarize our past research interests and provide some salient references; a complete list is provided in my full CV.
RNA structure – I have used NMR to study the basic structural elements of RNA, the building blocks that construct the most complex RNA structures, which at the time were unknown. As a post-doctoral fellow, I determined the structures of two of the most common building blocks of RNA (the tetraloop (1) and the E- or S-loop (2)) and have continued that work in Cambridge, where we interrogated the behavior of various other RNAs such as the HIV TAR stem-loop and genomic dimerization element, as well as various elements derived from human telomerase (in Seattle). A great recent source of satisfaction is our study of the GU wobble pair recognized so uniquely by the tRNAAla synthetase to achieve specific charging of tRNAAla: a just published structure in Nature showed that the mechanism we proposed was correct and even borrowed some of our sentences to explain their observations.
As the biological importance of non-coding RNA becomes ever more fully appreciated, we are now working on establishing the structure and mechanism of an RNA thermometer involved in the evasion of the immune response in N. meningitis; the structure of a paradigmatic and highly conserved long non coding RNA; and to establish how a promoter-associated long RNA recruits RNA-binding proteins to activate transcription of a proto-oncogene.
RNA-Protein Recognition – We were among the very first groups to study RNA-protein recognition using high-resolution structural methods (13), and have used NMR to determine the structures of RNA complexes derived from the two major classes of RNA-binding protein domains in eukaryotes (14-16). Several former students are now full professors at ETH, University College London and in Paris and still pursue this very activity with great success, fifteen or twenty years later. The structure of the U1A complex provided an example of highly specific RNA recognition by an RRM, the largest RNA-binding protein superfamily (3,17,18). It also provided insight into the regulation of gene expression through control of the status of the 3′-end of the mRNA, a very important process during development, differentiation and in the cellular response to growth or inflammatory signals. Technically, it represented a real tour de force, as a referee to the original publication correctly stated, and ushered an important area of structural biology, which has since grown a great deal. The structure of a double stranded RNA-binding domain (dsRBD) from Drosophila Staufen protein provided an almost opposite paradigm, since it represents an example of sequence-independent but structure-specific recognition of double-stranded RNA (4,19), and insight into RNA localization during early development and in the central nervous system.
The interest on U1A protein led us to study the structural biology of 3’-end processing (20-22) and its coupling with transcription (23,24) and RNA export/RNP assembly (25-29). More recently, we are studying how microRNA processing and maturation is regulated by RNA binding proteins, in no small part to generate the knowledge needed to inhibit these processes when mis-regulation results in human disease.
Structural biology of human disease – I wrote as early as 1991, when looking for my first faculty position, that there are tremendous opportunities to identify new inhibitors of the function of RNA and protein-RNA complexes with pharmaceutically attractive characteristics by using structure-based approaches (30,31). For example, regulation of transcriptional elongation of the HIV promoter involves an unusual RNA-dependent mechanism that provides unexploited targets for intervention. Thus, we determined the high-resolution structure of TAR RNA bound to peptides derived from Tat protein (7,32) and used this knowledge to discover peptidomimetic compounds that inhibit viral replication (10).
We pursued this discovery in a commercial context, when I co-founded a venture-capital supported biotechnology company: Ribotargets, now Vernalis on the London Stock Exchange. I have also been involved in another unsuccessful biotechnology start-up in Italy, on an unrelated scientific topic, kinases and matrix metallo-proteases. Much progress was made to discover small molecule inhibitors of the Tat-Tar interaction and of HCV translational control mechanisms (8,9), but the project was abandoned prior to pharmaceutical development because of a strategic decision on the part of the Board to abandon infectious diseases in favor of greater commercial opportunities in chronic conditions.
Since coming to Seattle, we have pursued peptidomimetic chemistry (33,34) and have recently identified compounds that specifically inhibit viral transactivation in cells and have antiviral activity within an order of magnitude of current antivirals (11,12,35). We have further improved the antiviral potency to levels better than those observed for AZT in primary cells by boosting binding activity to the picomolar range using rational design and gaining specificity at the same time. We are also using a technique developed in the biotechnology industry, NMR fragment-based screening, to identify new small molecule chemistry to target RNA (36).
Our future objective is to use the same approach to target critical RNA structures within microRNA precursors which are recognized by RNA-binding proteins to promote regulation of microRNA processing. By reducing the efficiency of processing of specific microRNAs, we will reduce over-expression of a specific set of microRNAs when this is correlated to cancer or inflammation.
The design of RNA-binding proteins – I have been fascinated by the problem of specificity in RNA-protein recognition for a long time (3,14-16,37): how can each of hundreds of essentially identical proteins belonging to the same structural class, for example the RRM, recognize different RNA sequences? We can continue mutating one or two amino acids or nucleotides at the time, measure thermodynamic characteristics, determine a structure or two, write a nice manuscript and start again with the next set of mutations or proteins. Or we might seek a paradigmatic shift by redesigning the specificity of a protein: if we can verify the prediction experimentally, we have answered the question of the origin of specificity in the scientifically most satisfying way. Furthermore, we would not only understand a biologically important and chemically intriguing problem, but will also generate new tools to probe gene expression pathways and alter gene expression.
Achieving this goal requires computational algorithms to design protein and RNA sequences and a way to score the energy of protein-RNA interfaces. We collaborated with Baker to demonstrate that protein design tools can effectively redesign a protein cores (38-41). In addition, we have defined two new scoring functions with different physical origin to evaluate the free energy of protein-RNA interfaces (42-44).
Despite the intellectual and technical challenges, which until now have not been successfully met by anyone, we have been able in recent years to re-target RNA-binding proteins of the RRM family to affect processing of non-cognate microRNAs in vitro and in cells. This is not just a tremendous technical achievement, but opens up new opportunities to interrogate and interfere with microRNA production and regulation.
Selected References
1. Cheong, C., Varani, G. and Tinoco, I., Jr. (1990) Solution Structure of an Unusually Stable RNA Hairpin, 5’GGAC(UUCG)GUCC. Nature, 346, 680-682.
2. Wimberly, B., Varani, G. and Tinoco, I., Jr. (1993) The Conformation of Loop E of Eukaryotic 5S Ribosomal RNA. Biochemistry, 32, 1078-1087.
3. Allain, F.-H.T., Gubser, C.C., Howe, P.W.A., Nagai, K., Neuhaus, D. and Varani, G. (1996) Specificity of Ribonucleoprotein Interaction Determined by RNA Folding during Complex Formation. Nature, 380, 646-650.
4. Ramos, A., Grunert, S., Adams, J., Micklem, D., Proktor, M., Bycroft, M., St Jhonston, D. and Varani, G. (2000) RNA Recognition by a Staufen Double-Stranded RNA Binding Domain. EMBO J., 19, 997-1009.
5. Mittermaier, A., Varani, L., Muhandiram, D.R., Kay, L.E. and Varani, G. (1999) Changes in Sidechain and Backbone Dynamics Identify Determinants of Specificity in RNA Recognition by Human U1A Protein. J. Mol. Biol., 294, 967-979.
6. Varani, G., Aboul-ela, F. and Allain, F.H.-T. (1996) NMR Investigations of RNA Structure. Progr. NMR Spectr., 29, 51-127.
7. Aboul-ela, F., Karn, J. and Varani, G. (1995) The Structure of the Human Immunodeficiency Virus Type-1 TAR RNA Reveals Principles of RNA Recognition by Tat Protein. J. Mol. Biol., 253, 313-332.
8. Collier, A.J., Gallego, J., Klinck, R., Cole, P.T., Harris, S.J., Harrison, G.P., Aboul-Ela, F., Varani, G. and Walker, S. (2002) A Conserved Structure within the HCV IRES eIF3 Binding Site Defines a New Antiviral Target. Nature Struct. Biol., 9, 375-380.
9. Davis, B., Afshar, M., Varani, G., Murchie, A.I.H., Karn, J., Lentzen, G., Drysdale, M., Bower, J., Potter, A.J., Starkey, I.D. et al. (2004) Rational Design of Inhibitors of HIV-1 TAR RNA through the Stabilisation of Electrostatic “Hot Spots”. J. Mol. Biol., 336, 343-356.
10. Hamy, F., Felder, E.R., Heizmann, G., Lazdins, J., Aboul-ela, F., Varani, G., Karn, J. and Klimkait, T. (1997) An Inhibitor of the Tat/TAR RNA Interaction that Effectively Suppresses HIV-1 Replication. Proc. Natl. Acad. Sci. USA, 94, 3548-3553.
11. Davidson, A., Leeper, T.C., Athanassiou, Z., Patora-Komisarska, K., Karn, J., Robinson, J.A. and Varani, G. (2009) Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein Proc. Natl. Acad. Sci. USA 106, 11931-11936.
12. Lalonde, M.S., Lobritz, M.A., Ratcliff, A., Chamanian, M., Athanassiou, Z., Tyagi, M., J., W., Robinson, J.A., Karn, J., Varani, G. et al. (2011) Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide tht binds the Tat-Transactivation response element (TAR) RNA. PLoS pathogens, 7, e1002038.
13. Howe, P.W.A., Nagai, K., Neuhaus, D. and Varani, G. (1994) NMR Studies of U1 snRNA Recognition by the N-Terminal RNP Domain of the Human U1A Protein. European Molecular Biology Organization Journal, 13, 3873-3881.
14. Varani, G. and Nagai, K. (1998) RNA Recognition by RNP Proteins during RNA Processing and Maturation. Ann. Rev. Biophys. Biomol. Struct., 27, 407-445.
15. Chen, Y. and Varani, G. (2005) Protein families and RNA recognition. FEBS J., 272, 2088-2097.
16. Lunde, B.M., Moore, C. and Varani, G. (2007) RNA-binding proteins: modular design for effcient function. Nature Rev. Mol. Cell. Biol., 8, 479-490.
17. Allain, F.H.-T., Howe, P.W.A., Neuhaus, D. and Varani, G. (1997) Structural Basis of the RNA Binding Specificity of Human U1A Protein. EMBO J., 16, 5764-5774.
18. Varani, L., Gunderson, S., Kay, L.E., Neuhaus, D., Mattaj, I. and Varani, G. (2000) The NMR Structure of the 38 kDa RNA-Protein Complex Reveals the Basis for Cooperativity in Inhibition of Polyadenylation by Human U1A Protein. Nature Struct. Biol., 7, 329-335.
19. Leulliot, N., Quevillon-Cheruel, S., Graille, M., van Tilbeurgh, H., Leeper, T.L., Godin, K.S., Edwards, T.E., Sigurdsson, S., Rozenkrants, N., Nagel, R.J. et al. (2004) A new a-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAseIII. The EMBO J., 23, 2468-2477.
20. Qu, X., Perez-Canadillas, J.-M., Agrawal, S., De Baecke, J., Cheng, H., Varani, G. and Moore, C. (2007) The C-terminal Domains of Vertebrate, CstF-64 and Its Yeast Orthologue Rna 15 Form a New Structure Critical for mRNA 3′ -End Processing. J. Biol. Chem., 282, 2101-2115.
21. Leeper, T.C., Qu, X., Lu, C., Moore, C. and Varani, G. (2010) Novel protein-protein contacts facilitate mRNA 3’-processing signal recognition by Rna15 and Hrp1. J. Mol. Biol.
22. Deka, P., Bucheli, M.E., Moore, C., Buratowski, S. and Varani, G. (2007) Structural Studies of the Interaction of the Yeast SR protein Npl3 with 3′-End Processing Sites. J. Mol. Biol. , 375, 136-150.
23. Perez-Canadillas, J.-M. and Varani, G. (2003) Recognition of GU-rich Polyadenylation Regulatory Elements by Human CstF-64 Protein. EMBO J, 22, 2821-2830.
24. Lunde, B., Reichow, S.L., Kim, M., Suh, H., Leeper, T.C., Yang, F., Mutschler, H., Buratowski, S., Meinhart, A. and Varani, G. (2010) Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nature Structural & Molecular Biology 17, 1195-1201.
25. Hamma, T., Reichow, S.L., Varani, G. and Ferre-D’Amare, A.R. (2005) The Cbf5-Nop10 complex is a molecular bracket that organizes box H/ACA RNPs. Nature Struct. Mol. Biol., 12, 1101-1107.
26. Reichow, S.L., Hamma, T., Ferra-D’Amare, A.R. and Varani, G. (2007) The structure and function of small nucleolar ribonucleoproteins. Nucleic Acids Res., 35, 1452-1464.
27. Leulliot, N., Godin, K.S., Hoareau-Aveilla, C., Quevillon-Cheruel, S., Varani, G., Henry, Y. and Van Tilbeurgh, H. (2007) The Box H/ACA RNP Assembly Factor Naf1p Contains a Domain Homologus to Gar1p Mediating its Interacion with Cbf5p. J. Mol. Biol. , 371, 1338-1353.
28. Godin, K.S., Walbott, H., Leulliot, N., van Tilbeurgh, H. and Varani, G. (2009) The Box H/ACA snoRNP assembly factor Shq1p is a chaperone protein homologous to Hsp90 cochaperones that binds to the cbf5p enzyme. J. Mol. Biol., 390, 231-244.
29. Wallbott, H., Machado-Pinilla, R., Liger, D., Blaud, M., Rety, S., Grozdanov, P.N., Godin, K., van Tilbeurgh, H., Varani, G., Meier, U.T. et al. (2011) The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic. Gene Dev, 25, 2398-2408.
30. Gallego, J. and Varani, G. (2001) Targeting RNA with Small Molecule Drugs: Therapeutic Promises and Chemical Challenges. Acc. Chem. Res., 34, 836-843.
31. Reichow, S.L. and Varani, G. (2006) RNA Switches Function. Nature, 441, 1054-1055.
32. Aboul-ela, F., Karn, J. and Varani, G. (1996) Structure of HIV-1 TAR RNA in the Absence of Ligands Reveals a Novel Conformation of the Trinucleotide Bulge. Nucleic Acids Res., 24, 3974-3981.
33. Athanassiou, Z., Dias, R.L.A., Moehle, K., Dobson, N., Varani, G. and Robinson, J.A. (2004) Structural Mimicry of Retroviral Tat Proteins by Constrained b-Hairpin Peptidomimetics – New Ligands with High Affinity and Selectivity for Viral TAR RNA Regulatory Elements. J. Am. Chem. Soc., 126, 6906-6913.
34. Leeper, T.C., Athanassiou, Z., Dias, R.L.A., Robinson, J.A. and Varani, G. (2005) TAR RNA recognition by a cyclic peptidomimetic of Tat protein. Biochemistry, 44, 12362-12372.
35. Davidson, A., Patora-Komisarska, K., Robinson, J.A. and Varani, G. (2011) Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein. Nucleic Acids Research, 39, 248-256.
36. Davidson, A., Begley, D.W., Lau, C. and Varani, G. (2011) A Small-Molecule Probe Induces a Conformation in HIV TAR RNA Capable of Binding Drug-Like Fragments. Journal of Molecular Biology, 410, 984-996
37. Leulliot, N. and Varani, G. (2001) Current Topics in RNA-Protein Recognition: Control of Specificity and Biological Function through Induced Fit and Conformational Capture. Biochemistry, 40, 7947-7956.
38. Kuhlman, B., Dantas, G., Ireton, G.C., Varani, G., Stoddard, B.L. and Baker, D. (2003) Design of a Novel Globular Protein Fold with Atomic-Level Accuracy. Science, 302, 1364-1368.
39. Dobson, N., Dantas, G., Baker, D. and Varani, G. (2006) High-Resolution Structural Validation of the Computational Redesign of Human U1A Protein. Structure, 14, 847-856.
40. Dantas, G., Corrent, C., Reichow, S.L., Havranek, J.J., Eletr, Z.M., Isern, N.G., Kuhlman, B., Varani, G., Merritt, E.A. and Baker, D. (2007) High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design. J. Mol. Biol., 366, 1209-1221
41. Watters, A.L., Deka, P., Corrent, C., Callender, D., Varani, G., Sosnick, T. and Baker, D. (2007) The Highly Cooperative Folding of Small Naturally Occurring Proteins is Likely the Results of Natural Selection. Cell, 128, 613-624
42. Robertson, T.A. and Varani, G. (2007) An All-Atom, Distance-Dependent Scoring Function for the Prediction of Protein-DNA Interactions From Structure. Proteins: Struct. Func. Gen., 66, 359-374.
43. Zheng, S., Robertson, T.A. and Varani, G. (2007) A knowledge-based potential function predicts the specificity and relative binding energy of RNA-binding proteins. FEBS Journal, 274, 6378-6391.
44. Chen, Y., Mandic, J. and Varani, G. (2008) Cell-free selection of RNA-binding proteins using in vitro compartmentalization. Nucleic Acids Res., 36.