RNA silencing, also known as RNA interference, is a conserved biological response to double-stranded RNA that regulates gene expression. The response is mediated by small interfering RNAs (siRNAs), which guide the sequence-specific degradation of cognate messenger RNAs (mRNAs). Our long-term goals are to structurally characterize and mechanistically define events associated with (1) processing of long double-stranded RNAs into siRNAs by the endonuclease acvtivity of Dicer and (2) guide-strand-mediated cleavage of target RNAs by Argonaute, the key component exhibiting slicer activity, within the RNA-induced silencing complex (RISC). We are also interested in (3) protein-RNA complexes along the microRNA (miRNA) biogenesis pathway that mediate processing of primary miRNAs to their precursor counterparts, and processes associated with miRNA guide strand-mediated cleavage, translation inhibition or degradation of target RNAs. This research involves a collaborative effort with the Thomas Tuschl laboratory at The Rockefeller University.
Our laboratory has published the following review on RNA silencing:
Patel, D. J., Ma, J-B., Yuan, Y-R., Ye, K., Pei, Y., Kuryavyi, V., Malinina, L., Meister, G. & Tuschl, T. (2007). Structural biology of RNA silencing and its functional implications. Cold Spring Harbor Laboratory Symposium on Regulatory RNAs 71, 81-93. [PubMed Abstract]
Argonaute-Mediated Target RNA Cleavage
Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC), contributing to both the architectural and catalytic functionalities associated with siRNA guide-strand selection within the RISC loading pathway, and subsequent guide-strand-mediated cleavage of complementary mRNA by catalytically competent RISC. We have undertaken structure-function studies to define the domain architecture of bacterial Agos, identify the nucleic-acid-binding channel that accommodates the bound guide strand, define the pairing between guide and target in the ternary complex, and elucidate the mechanistic basis for site-specific target RNA cleavage. In addition, we are interested in defining the conformational transitions within Ago during the various steps of the catalytic cleavage cycle.
Ternary Argonaute Silencing Complex Containing Guide-Target Duplex Spanning the Seed Segment
We have solved the 3.0 Å crystal structure of a ternary complex of wild-type T. thermophilus Ago bound to a 5'-phosphorylated 21-mer guide DNA and a 20-mer target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released upon ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of Ago widens on formation of a more open ternary complex. The relationship of structure to function was interrogated in the Thomas Tuschl laboratory by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge, and 2'-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing within the seed segments of the target, but not the guide strand, were better accommodated and their complexes catalytically active.
Wang, Y., Li, H., Juranek, S., Sheng, G., Tuschl, T. & Patel, D. J. (2008). Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921-926. [PubMed Abstract]
Guide-Strand-Containing Binary Argonaute Silencing Complex
We have solved the crystal structures of Aa-Ago bound to 22-mer and 26-mer siRNAs, where we have unexpectedly identified externally bound Ago-siRNA complexes. One 2-nt 3'-overhang of the siRNA inserts into a channel positioned on the outer surface of the PAZ-containing lobe of the bilobal Aa-Ago architecture. The first overhang nucleotide stacks over a conserved tyrosine ring, while the second overhang nucleotide, together with the intervening sugar-phosphate backbone, inserts into a preformed surface channel. Photochemical cross-linking studies of Aa-Ago with 5-iodo-U-labeled ssRNA and siRNA undertaken in our collaborator Thomas Tuschl's laboratory, provide support for this externally bound Ago-siRNA complex, with the structural and cross-linking results together providing insights into a protein-RNA recognition event potentially associated with the RISC-loading pathway.
Yuan, Y. R., Pei, Y., Chen, H. Y., Tuschl, T. & Patel, D. J. (2006). A potential protein-RNA recognition event along the RISC-loading pathway from the structure of A. aeolicus Argonaute with externally bound siRNA. Structure 14, 1557-1565. [PubMed Abstract]
Bacterial Argonautes Are DNA Guide-Strand-Mediated Site-specific Endoribonucleases
The Piwi protein is composed of two domains, Mid and PIWI, with the latter shown previously to adopt an RNase H fold critical for the endoribonuclease cleavage activity of the RNA-induced silencing complex (RISC). Our group, and that of David Barford at the Institute of Cancer Research, London, have solved the crystal structure of Archaeoglobus fulgidus Piwi protein bound to siRNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated mRNA target cleavage specificity. The phosphorylated 5'-end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the C-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5'-end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a short RNA duplex. Mutation studies in the Thomas Tuschl laboratory of the corresponding amino acids that contact the 5'-phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-siRNA complex provides direct support for the 5'-region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5'-end of the guide RNA.
Ma, J. B., Yuan, Y. R., Meister, G., Pei, Y., Tuschl, T. & Patel, D. J. (2005). Structural basis for 5'-end-specific recognition of the guide RNA strand by the A. fulgidus PIWI protein. Nature 434, 666-670. [PubMed Abstract]
siRNA 2-nt 3'-Overhang End Recognition by the PAZ Domain
As a counter-defense strategy to host immunity directed by small RNAs, genetically diverse plant and animal viruses encode numerous viral suppressors of RNA silencing (VSRs). The plant cucumoviral 2b protein is among the best-characterized VSRs for the activity to suppress non-cell autonomous RNA silencing. We have collaborated with the Shon-Wei Ding laboratory at the University of California, Riverside, to show that 2b from Tomato Aspermy Virus (Tav2b) binds to both siRNA and long dsRNA and report the crystal structure of Tav2b (1-69) in complex with a 21-nt siRNA duplex. Remarkably, the major groove of the fully complementary siRNA duplex is dramatically widened and deeply penetrated by two long á-helices of Tav2b, with complex formation stabilized by non-sequence-specific intermolecular interactions between basic amino acid side chains and RNA backbone phosphates. The structure of the Tav2b-siRNA complex highlights novel structural scaffolds and recognition principles for dsRNA binding and reflects an alternative evolutionary adaptation strategy developed by viruses to overcome the RNA-silencing immunity of their hosts.
Ma, J. B., Li, F., Li, H-W., Li, W-X., Ding, S-W. & Patel, D. J. (2009). Structural basis for siRNA recognition by a viral suppressor of non-cell autonomous RNA silencing.
Viral p21 Protein
The p19 protein from the tombusvirus is a viral suppressor of RNA silencing and has been shown to bind specifically to siRNA. Our group, and that of Traci Tanaka-Hall at NIEHS, have independently solved the crystal structure of p19 bound to a 21-nt siRNA, where the 19-bp RNA duplex is cradled within the concave face of a continuous eight-stranded b-sheet, formed across the p19 homodimer interface. Direct and water-mediated intermolecular contacts are restricted to the backbone phosphates and sugar 2'-OH groups, consistent with sequence independent p19-siRNA recognition. Two a-helical “reading heads” project from opposite ends of the homodimer and position their pairs of tryptophans for stacking over the terminal base pairs, thereby measuring and bracketing both ends of the siRNA duplex. Our structure provides an illustration of siRNA sequestration by an evolved viral protein.
Ye, K., Malinina, L. & Patel, D. J. (2003). Recognition of siRNA by a viral suppressor of RNA silencing. Nature 426, 874-878. [PubMed Abstract]