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 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.
Recognition of siRNA Ends
Three critical features of siRNAs are their length, the 2-nt overhang at the 3'-end and the phosphate at the 5'-end. Our group has attempted to understand how proteins recognize these unique features of siRNAs.
The PAZ domain is an RNA-binding module found in Argonaute (Ago) and some Dicer proteins and its structure has been determined previously in the free state. We have solved the crystal structure of the PAZ domain from human Ago eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence independent manner, PAZ anchors the 2-nt 3'-overhang of the siRNA-like duplex within a highly conserved binding pocket. It further secures the duplex by binding the 7-nt phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an 3'-end binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3'-end of guide RNA within silencing effector complexes.
Ma, J-B., Ye, K. & Patel, D. J. (2004). Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318-322.
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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 provide 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.
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.
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Our group, and that of Leemor Joshua-Tor at the Cold Spring Harbor Laboratory, have independently solved crystal structures of bacterial Agos in the free state. Our Aquifex aeolicus (Aa) Ago structure adopts a bilobal architecture composed of N and PAZ-containing and Mid and PIWI-containing lobes. Binding and cleavage studies in collaboration with the Thomas Tuschl laboratory establish this eubacterial Ago to be a guide DNA strand-mediated site-specific endoribonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5'-phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by the RNase H type cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments in the Tuschl laboratory involving chimeras of human Ago1 and cleavage competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We have proposed a four-step Ago-mediated catalytic cycle cleavage model, which provides distinctive perspectives into the mechanism of guide strand-mediated mRNA cleavage within RISC.
Yuan, Y. R., Ma, J. B., Kuryavyi, V., Pei, Y., Zhadina, M., Meister, G., Chen, H. Y., Dauter, Z., Tuschl, T. & Patel, D. J. (2005). Crystal structure of Aquifex aeolicus Argonaute provides unique perspectives into the mechanism of guide strand-mediated mRNA cleavage. Mol. Cell 19, 405-419.
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 undertakenin 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.
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The slicer activity of the RNA induced silencing complex (RISC) is associated with argonaute (Ago), whose RNase H-like PIWI domain catalyzes guide-strand-mediated sequence specific cleavage of target message. We have solved the crystal structure of T. thermophilus Ago bound to a 5'-phosphorylated 21-mer DNA guide strand, thereby identifying the nucleic acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, while two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies in the Thomas Tuschl laboratory indicate that key amino acid residues at the active site and the Mid domain binding pocket are critical for cleavage activity, while alterations at the PAZ domain binding pocket show little effect.
Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. (2008). Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209-213.
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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.
Viral Suppressors of Silencing
Many viruses encode proteins that specifically inhibit the RNA silencing machinery, thereby protecting the viral mRNA from degradation. Our group has a research program focused on the structural characterization of the complexes of viral suppressors with their RNA and protein targets.
The p19 protein from the tombusvirus is such 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.
Beet yellow virus (BYV) is a positive strand virus that features a large genome and long filamentous virions. p21 is the only protein in the BYV genome that has been shown to suppress RNA silencing. Our crystal structure of p21 in the free state has established that the protein adopts an octameric ring architecture, with a large central cavity of 90 Å diameter. The all a-helical p21 monomer consists of N- and C-terminal domains that associate with their neighboring counterparts through symmetric head-to-head and tail-to-tail interactions. A putative RNA-binding surface has been identified in the conserved, positive-charged inner surface of the ring. In contrast to the specific p19-siRNA duplex interaction, p21 is a general nucleic acid bonding protein targeting both single and double-stranded, independent of length.
Ye, K. & Patel, D. J. (2005). RNA silencing suppressor p21 of beet yellow virus forms an RNA-binding octameric ring structure. Structure 13, 1375-1384.
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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 α-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.
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