Short RNAs, as regulators of cellular function, can impact on the maintenance of genomic integrity and stability, on cell growth, differentiation and developmental processes, and on the antiviral RNA silencing response. RNA silencing refers to small interfering RNA (siRNA)-mediated post- transcriptional gene regulation, resulting in the silencing of viral genes and transgenes. Such host- virus interactions involve highly specific, adaptive, mobile and systemic processes that operate in essence as a RNA-based immune response. siRNAs, made up of 19 to 23 base pair duplexes, with 2-nt S'-overhangs and 5'-phosphates, as part of the RNA-induced silencing complex (RISC), target complimentary viral mRNAs and tag them for degradation. Viruses evolve silencing suppressor proteins to counteract RNA silencing by modifying the accumulation and/or the activity of siRNAs associated with the anti-viral response. This application proposes to continue our ongoing structure-function studies of protein-RNA complexes associated with RISC-mediated RNA silencing, as well as silencing suppression by evolved viral suppressor proteins. Our group has already solved the crystal structures of human PAZ-siRNA and A. fulgidus Piwi-siRNA complexes, of A. aeolicus Argonaute (Ago) in the free and externally siRNA-bound states, as well as the viral suppressors p19 and p21 in the siRNA-bound and free states, respectively. These structures provide a framework for defining additional experiments to decipher the functional states and conformational transitions of bacterial and human Ago's during binding, processing and release of guide RNA and mRNA associated with the RISC-mediated catalytic cycle. It is our expectation that the proposed structural-mutational-functional studies undertaken in collaboration with the Thomas Tuschl laboratory at Rockefeller University will provide unparalleled insights into mechanistic issues associated with individual steps of the RNA silencing pathway. Our initial studies of protein- RNA recognition on the siRNA-binding p19 viral suppressor are being extended to the p21 suppressor, which adopts an octameric ring architecture. Such studies should eventually provide a solid foundation for developing approaches that achieve developmental-stage or tissue-specific expression of viral suppressors, thereby inhibiting cell-specific silencing pathways in plants and animals.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Park, Eun-Chung
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Sloan-Kettering Institute for Cancer Research
New York
United States
Zip Code
Liu, Yiwei; Esyunina, Daria; Olovnikov, Ivan et al. (2018) Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA. Cell Rep 24:453-462
Tian, Yuan; Simanshu, Dhirendra K; Ma, Jin-Biao et al. (2014) A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer. Mol Cell 53:606-16
Sheng, Gang; Zhao, Hongtu; Wang, Jiuyu et al. (2014) Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proc Natl Acad Sci U S A 111:652-7
Nakanishi, Kotaro; Ascano, Manuel; Gogakos, Tasos et al. (2013) Eukaryote-specific insertion elements control human ARGONAUTE slicer activity. Cell Rep 3:1893-900
Nakanishi, Kotaro; Weinberg, David E; Bartel, David P et al. (2012) Structure of yeast Argonaute with guide RNA. Nature 486:368-74
Park, Jong-Eun; Heo, Inha; Tian, Yuan et al. (2011) Dicer recognizes the 5' end of RNA for efficient and accurate processing. Nature 475:201-5
Weinberg, David E; Nakanishi, Kotaro; Patel, Dinshaw J et al. (2011) The inside-out mechanism of Dicers from budding yeasts. Cell 146:262-76
Tian, Yuan; Simanshu, Dhirendra K; Ascano, Manuel et al. (2011) Multimeric assembly and biochemical characterization of the Trax-translin endonuclease complex. Nat Struct Mol Biol 18:658-64
Tian, Yuan; Simanshu, Dhirendra K; Ma, Jin-Biao et al. (2011) Structural basis for piRNA 2'-O-methylated 3'-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains. Proc Natl Acad Sci U S A 108:903-10
Wang, Yanli; Ludwig, Janos; Schuberth, Christine et al. (2010) Structural and functional insights into 5'-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat Struct Mol Biol 17:781-7

Showing the most recent 10 out of 14 publications