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). 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. 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 Symposium Laboratory on Regulatory RNAs, 71, 81-93.
Recognition of siRNA Ends
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.
Viral Suppressors of Silencing
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Many viruses encode proteins that specifically inhibit the RNA silencing machinery, thereby protecting the viral mRNA from degradation. 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.
Role of Argonaute Proteins in the RNA Induced Silencing Complex
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.
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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 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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