PHS 398 (Rev. 06/09), Biographical Sketch Format Page

NAME POSITION TITLE Gabriele Varani Professor of Biochemistry and Professor of Chemistry INSTITUTION AND LOCATION DEGREE MM/YY FIELD OF STUDY Laurea (cum Universita’ di Milano 10/1982 Physics laude) Universita’ di Milano Doctorate 10/1987 Physics/Biophysics Post- University of California Berkeley 1987-1992 Structural Biology doctoral

A. Personal Statement Our primary research interest lies in studies of RNA- and RNP--structure function; their role in human disease; and in targeting RNA with small molecule, peptides and proteins. The project described in this proposal emerges from our long term interests in the molecular basis of protein-RNA recognition and RNA structure, and in targeting RNA with small molecule, peptides and proteins for pharmacological applications. I have been working on the structural biology of small molecule binding to RNA and of RNA-binding proteins since the early 1990s, when I started as a faculty at the MRC in Cambridge. We determined some of the very first high resolution structures of RNAs and of RNA-protein complexes and became interested in the structural biology of HIV in the early 1990s and of RNA processing about 20 years ago. Since 1992, I have been interested in discovering small molecules of pharmacological value that bind to RNA, and have pursued this interest in Academia, in a successful start-up and in various collaborations with big pharma and the biotech industry. We have used NMR and x-ray crystallography to determine structures of proteins, protein-RNA and protein-protein complexes involved in these important biological processes. We use primarily NMR as a tool for structural biology of individual proteins, RNA and simple protein-RNA and protein-protein complexes, and apply crystallography or electron microscopy (EM) to complexes that are beyond the size of NMR studies. We run assays for binding in a variety of formats (biochemically and in cell lines) and characterize the structure of RNA complexes with proteins, peptides and small molecules using NMR. We conduct biochemical investigations of the function of the proteins and RNAs we study, and collaborate with molecular biologists, virologists and cancer biologists to provide in vivo models and biological context to our structural and biophysical work.

A complete list of my publications (with citations) can be found on google scholar. Publications most relevant to the present project can be found in section C.4 and C.3. My bibliography includes 181 peer-reviewed publications as of 12-31-2015; for whatever is worth, my H-factor is 58. B. Positions and Honors Positions and Employment MRC-Laboratory of Molecular Biology, Cambridge, England, Group Leader (tenure-track), 1992-1996 MRC-Laboratory of Molecular Biology, Cambridge, England, Senior Scientist (tenured), 1996-2001 Department of Biochemistry and Department of Chemistry University of Washington, Professor, 2001-present

Honors La Riccia Foundation Post-Doctoral Fellow, 1987-1989 Visiting Professor, Universita’ di Roma, 2000-2001 Gilead Lecture, Sheffield 200; AAAS Newcomb Cleveland Prize, co-author of best article in Science, 2004 >200 invited seminars and lectures at international meetings and universities.

Professional activities Editorial Board, Nucleic Acid Research 1998-2001 Associate Editor, European Journal of Biochemistry 2003-2013 Member of MRC Reviewer Board 1998-2002 Service: NSF Scientific instrumentation panel, 2004; NIH Special Emphasis panel 2004; MFSB 2005, 2007; MFSD 2008; MFSC 2011; fellowships panel 2006, EBT 2009, BCMB-K 2010, UCSD PO1 Review panel 2010, NIH Instrumentation panel 2010, MSFC 2011; Harvard NMR Center PO1 2012; ADDT Special Emphasis panel 2013; AIDSRRC 2013 and 2014, MSFC 2015; MSFD 2015-2021 permanent member Scientific Advisory Board, Pittsburgh Center for HIV Protein Interactions 2008-; RNA Center at SUNY Albany 2013-; NMRFAM at Madison 2013- Consultancies Founder and Scientific Advisory Board, Vernalis 1997-2001 Consultant, Allelix 1994-1995; CIBA Geigy 1994; Parke Davis 1995; Groove Pharmaceuticals, 2011-2012

C. Contributions to science 1. Binding of drug-like small molecules to RNA I wrote in 1991, applying for my first faculty position, that there are tremendous opportunities to identify new inhibitors of the function of RNA and protein-RNA complexes in bacteria and viruses and that structure-based rational approaches would provide insight into how to target RNA with small drug-like molecules (Ref C.1.4). We pursued RNA transcription and translation regulatory elements in HIV (see item 2 below) and the hepatitis C virus (C.1.3). 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. We participated in the design of small molecules (<350 Da) with Lipinski-rule pharmacological characteristics that bound to RNA with low nM activity (C.1.2). In an effort to investigate the role of RNA in chronic disease, we studied RNA regulatory elements that control splicing of mRNA involved in neurodegenerative disease (C.1.1). We exploits these same ideas to target microRNA precursors which are recognized by RNA-binding proteins to promote regulation of microRNA processing. By reducing the efficiency of processing of specific microRNAs, such as miR-21, we will reduce its over-expression in cancer. 1. S. Zheng, Y. Chen, C. Donahue, M. S. Wolfe and G. Varani Structural basis for stabilization of the tau pre-mRNA splicing regulatory element by novatrone (mitoxantrone) Chem Biol 16 557-566 (2009) (with News and Views). PMCID: PMC2759301 (42 cits) 2. 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 Stabilization of Electrostatic "Hot Spots". J. Mol. Biol., 336, 343-356 (103 cit.) 3. 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 (110 citations) 4. Gallego, J. and Varani, G. (2001) Targeting RNA with Small Molecule Drugs: Therapeutic Promises and Chemical Challenges. Acc. Chem. Res., 34, 836-843 (242 cit.)

2. Peptide mimetics to inhibit RNA function in disease Regulation of transcriptional of the HIV promoter involves an RNA-dependent mechanism that provides unexploited targets for antiviral intervention. We determined the high-resolution structure of TAR RNA bound to peptides derived from Tat protein (ref C.2.5) and used this knowledge to discover peptidomimetic compounds that inhibit viral replication (C.2.3 and C.2.4). We further pursued diverse peptidomimetic chemistry at the University of Washington (C.2.1; C.2.2) and have recently identified compounds that specifically inhibit viral transactivation in cells and have antiviral activity comparable to AZT. We have further improved the antiviral potency to levels better than those observed for AZT in primary cells by boosting binding to 30 picomolar using rational design (Jones et al, under review J. Am. Chem. Soc.). Our experience of >20 years indicates that this scaffold provides by far the best binding capacity for RNA we have so far encountered. 1. M. S. Lalonde, M. A. Lobritz, A. Ratcliff, Z. Athanassiou, Mudit Tyagi, J. A. Robinson, J. Karn, G. Varani and E. J. Arts Dual inhibition of HIV-1 reverse transcription and mRNA transcription by a conformationally constrained peptidomimetic that binds the Tat-transactivating response element (TAR) in HIV-1 genomic RNA PLOS Pathogens (2011) PMCID: PMC3098202 (31 cits) 2. A. Davidson, T. C. Leeper, Z. Athanassiou, K. Patora-Komisarska, J. Karn, J. A. Robinson and G.Varani Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein PNAS USA 106 11931-11936 (2009). PMCID: PMC2715490 (85 cits) 3. Z. Athanassiou, R. L. A. Dias, K. Moehle, N. Dobson, G. Varani and J. A. Robinson Structural Mimicry of Retroviral Tat Proteins by Constrained b-Hairpin Peptidomimetics: Ligands with High Affinity and Selectivity for Viral TAR RNA Regulatory Elements J. Am. Chem. Soc. 126 6906-6913 (2004) (61 cits) 4. 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 (259 cits.) 5. 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 (391 cit.) 3. Structure of RNA-protein complexes and their function in gene expression We were one of the very first groups to study RNA-protein recognition using high-resolution structural methods. Former students who participated in these early efforts are now full professors at ETH, University College London and in Paris and still pursue this activity with great success. The structure of the U1A complex provided an example of highly specific RNA recognition by an RRM, the largest RNA-binding protein superfamily (ref C.3.1). 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 stated. The structure of a double stranded RNA-binding domain (dsRBD) from Staufen protein provided the opposite paradigm, since it represents an example of structure-specific recognition of double-stranded RNA, and insight into RNA localization during early development and in the central nervous system (C.3.2). The interest on U1A protein led us to study the structural biology of 3’-end processing (C.3.3) and its coupling with transcription and RNA export/RNP assembly (C.3.4). More recently, and relevant to the current project, we are studying how microRNA processing and maturation is regulated by RNA binding proteins (Y. Chen et al, under review). 1. 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 (261 cits.) 2. 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 (295 cits.) 3. 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 (130 cits) 4. 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 PMCID: PMC2050884 (70 cits.)

4. The molecular basis of protein-RNA recognition and the design of RNA-binding proteins I have been fascinated by the problem of specificity in RNA-protein recognition for a long time. As discussed in an often cited review (Ref C.4.1): 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, 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 alter gene expression. We collaborated with Baker to demonstrate that protein design tools can effectively redesign a protein cores in a true landmark manuscript (C.4.2). We defined multiple computational approaches to evaluate the free energy of protein-nucleic acid complexes (C.4.3; C.4.4), which are still imitated (with increasing success given the greater data set of structures on which they are based). We have been able in recent years to re-design an RRM to affect processing of non-cognate microRNAs in vitro and in cells (Y. Chen et al, under review, Nature Chem. Bio.). This is in my judgment (and the referees) a breakthrough, which opens up new opportunities to interrogate and interfere with microRNA production and regulation. 1. 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 (455 cits) 2. 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 (>1,000 cits) 3. 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 (55 cit.) 4. Y. Chen, T. Kortemme, T. Robertson, D. Baker and G. Varani, A new hydrogen-bonding potential for the design of protein-RNA interactions predicts specific contacts and discriminated decoys. Nucleic Acids Res. 32 5147-5162 (2004) (60 cits) 5. Structure and function of RNA I used NMR to study the building blocks that construct the most complex RNA structures, which at the time were unknown. Some of this work, considered classical, has been cited hundreds of times (C.5.1; C.5.2). I determined the structures of two of the most common building blocks of RNA (the tetraloop and the E- or S- loop), and 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 and riboswitches (C.5.4). A 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 (C.5.3): a structure published in Nature in 2014 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 (R. Barnwal et al, under review) and of several ncRNAs. 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 (428 cits) 2. Wimberly, B., Varani, G. and Tinoco, I., Jr. (1993) The Conformation of Loop E of Eukaryotic 5S Ribosomal RNA. Biochemistry, 32, 1078-1087 (261 cits) 3. A. Ramos and G. Varani Structure of the Acceptor Stem of E.coli tRNAAla: Role of the G3-U70 Base pair in Synthetase Recognition Nucleic Acids Res. 25 2083-2090 (1997) (108 cits) 4. M.-K. Lee, M. Gal, L. Frydman and G. Varani Real-time multidimensional NMR folding the adenine- sensing riboswitch with second resolution PNAS 107 9192-9197 (2010) PMCID: PMC2889053 (59 cits)

D. Mentoring The PI has trained in the past 16 graduate students, 16 post-doctoral fellows and >20 undergraduate students. The researcher teaches: Chemistry 453 once per year (senior level physical chemistry for biochemistry majors); Chemistry 152 once per year (part of the freshman chemistry series). He has taught for >10 years Biochemistry 530 conjoint (first year graduate biophysical methods); Biochemistry 540 (first year graduate writing course) and Biochemistry 534 (every 2 years, graduate NMR course). He currently supervises 3 graduate students, two of whom are URM’s and/or from rural economic disadvantaged populations, 8 post- docs and senior scientists and 6 undergraduate students; 3 of whom are URMs. Dr. Varani has served in various capacities in the Recruitment and Steering committees for the Molecular Biophysics Training Grant and both the Biochemistry and Chemistry Departments. Dr. Varani has been actively involved in education of under-represented minorities (URMs), and has a very strong professional interest and personal commitment to education of underrepresented minorities. He has done so as a mentor to multiple undergraduate students, as member of the Faculty Advisory Board of IMSD (Initiative to Maximize Student Diversity); he is now the director of UW-PREP, a post-baccalaurate (PREP) program, where Dr Varani is also the PI; he has also been a member of the Health Science Center Minority Center Advisory Board and of the Diversity Council at the University of Washington.