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.

Our laboratory has published the following review on RNA silencing:

Swarts, D. C., Makarova, K., Wang, Y., Nakanishi, K., Ketting, R. F., Koonin, E. V., Patel, D. J. and van der Oost, J. (2014). The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743-753.

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]

Dicer-Mediated dsRNA Cleavage

RNase III enzymes play central roles in RNA silencing by processing double-stranded RNA precursors into small RNA duplexes. Drosha cleaves primary miRNA (pri-mRNA) to release a hairpin-shaped precursor miRNA (pre-miRNA). Dicer cuts the pre-miRNA near the terminal loop and generates a short miRNA duplex. Dicer also participates in small interfering RNA (siRNA) production from long RNA duplexes. One strand of the small RNA duplex is subsequently loaded onto the Argonaute protein to yield an active RNA-induced silencing complex. Precise selection of cleavage sites by RNase III enzymes is critical, with Drosha and Dicer recognizing specific RNA structures and cleave a fixed distance away from that structural element.

A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer

A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer

We have solved two families of crystal structures of human Dicer ‘platform-PAZ-connector helix’ cassette in complex with siRNAs as part of a collaborative effort with the Narry Kim laboratory (Seoul National University, South Korea). The structures possess two adjacently positioned pockets: a 2-nucleotide 3’-overhang-binding pocket within the PAZ domain (3’-pocket) and a newly identified phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contain a knob-like a-helical protrusion, designated ‘hDicer-specific helix’, that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned towards the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5’-phosphate from the phosphate pocket, while retaining the 3’-overhang in the 3’-pocket.

Tian, Y., Simanshu, D. K., Ma, J. B., Park, J-E, Heo, I., Kim, V. N. & Patel, D. J. (2014). A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer. Mol. Cell 53, 606-616.

Dicer Recognizes the 5’-end of RNA for Efficient and Accurate Processing

Dicer Recognizes the 5’-end of RNA for Efficient and Accurate Processing

A hallmark of RNA silencing is a class of ~22 nt RNAs which are processed from dsRNA precursors by Dicer. Accurate processing by Dicer is critical for the functionality of microRNAs (miRNAs). According to the current model, Dicer selects the cleavage sites by measuring a set distance from the 3’ overhang of the dsRNA terminus. Here the Narry Kim laboratory (Seoul National University) has championed studies that establish that human Dicer anchors not only the 3’ end but also the 5’ end, with the cleavage site determined mainly by the distance (~22 nt) from the 5’ end (5’ counting rule). This mode of cleavage requires a 5’-terminal phosphate group. We further identified from structural studies a novel basic motif (5’-pocket) in human Dicer, for the recognition of the 5’-phosphorylated end. The 5’ counting rule and the 5’-anchoring residues are conserved in miRNA-generating enzymes such as Drosophila Dicer-1 but not in Giardia Dicer. Mutations in the 5’-pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5’-pocket mutant. Thus, the 5’-phosphorylated end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.

Park, J. E., Heo, I., Tian, Y., Simanshu, D. K., Chang, H., Jee, D., Patel, D. J. & Kim, V. N. (2011). Dicer recognizes the 5’-end of RNA for efficient and accurate cleavage. Nature 475, 201-205.

Inside-Out Mechanism of Dicers from Budding Yeasts

Inside-Out Mechanism of Dicers from Budding Yeasts

The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeast. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses undertaken in the laboratory of our collaborator David Bartel (MIT and Whitehead Institute) showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural protein-based molecular rulers and imparts substrate preferences with ramifications for biological function.

Weinberg, D., Nakanishi, K., Patel, D. J. & Bartel, D. P. (2011). The inside-out mechanism of Dicers from budding yeasts. Cell 146, 262-276.

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 eubacterial 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. The structural studies on eubacterial Argonaute proteins have recently been extended to their eukaryotic counterparts, mainly budding yeast and human Argonautes.

Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity

Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity

Towards understanding the structural and biochemical differences in human ARGONAUTE (hAGO) RNA-guided RNA cleavage activity, we solved the crystal structure of cleavage-inactive hAGO1 bound to 5’-phosphorylated guide RNA and compared it with previously published structures of cleavage-active hAGO2 counterparts. A conserved insertion element, termed the cS7 loop previously identified in the structure of hAGO2, is kinked due to prolines at positions 670 and 675 within hAGO1, thereby forming a convex surface that is juxtaposed with the nucleic-acid-binding channel near the catalytic site. As such, cS7 appears to sterically hinder guide-target RNA pairing, rather than directly affecting the catalytic reaction. Biochemical assays in the laboratory of our collaborator Thomas Tuschl (Rockefeller University) established that the P675Q substitution in the presence of the DEDH reconstituted catalytic tetrad led to hAGO1 cleavage activity. Combined substitutions of R805H in the catalytic pocket and P675Q in cS7 were sufficient to convert hAGO1 into a partially active enzyme, highlighting a structural role for the cS7 in modulating miRNA guide-dependent cleavage.

Nakanishi, K., Ascano, M., Gogakos, T., Ishibi-Murakami, S., Serganov, A. A., Briskin, D., Morozov, P., Tuschl, T. and Patel, D. J. (2013). Eukaryote-specific insertion elements control human ARGONAUTE slicer activity. Cell Reports 3, 1893-1900.

EGFR Modulates MicroRNA Maturation in Response to Hypoxia Through Phosphorylation of AGO2

In a collaborative project championed by the Mien-Chie laboratory (M. D. Anderson Cancer Center), it has been shown that epidermal growth factor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific- tumor-suppressor-like miRNas in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients.

Shen, J., Xia, Y., Khotskaya, Y. B., Huo, L., Nakanishi, K., Lim, S-O., Du, Y., Wang, Y., Chang, W-C., Chen, C-H., Hsu, J. L., Lam, Y. C., James, B. P., Liu, C-G., Liu, X., Patel, D. J. & Hung, M. C. (2013). EGFR modulates miRNA maturation in response to hypoxia through phosphorylation of Ago2. Nature 497, 383-387.

Structure of Yeast Argonaute with Guide RNA

Structure of Yeast Argonaute with Guide RNA

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here in collaboration with the David Bartel laboratory (MIT and Whitehead Institute), we report the 3.2 Å crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide RNA nucleotides 1 to 8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2’-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in near A-form conformation. Compared with prokaryotic Argonautes, KpAgo has numerous surface exposed insertion elements, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAgo has a hydrogen-bonded network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.

Nakanishi, K., Weinberg, D. E., Bartel, D. P. & Patel, D. J. (2012). Structure of yeast Argonaute with guide RNA. Nature 486, 368-374.

DNA-guided DNA interference by a prokaryotic Argonaute

A project championed by the John van der Oost laboratory (Wageningen University, The Netherlands) has demonstrated that the Argonaute of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5’-phoshorylated DNA guides, 13-25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5’-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Thus, TtAgo functions in host defense by DNA-guided DNA interference.

Swarts, D. C., Jore, M. M., Westra, E. R., Zhu, Y., Janssen, J. H., Snijders, A. P., Wang, Y., Patel, D. J., Berenguer, J., Brouns, S. J. and van der Oost, J. (2014). DNA-guided DNA interference by prokaryotic Argonaute. Nature 507, 258-261.

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5’-phosphorylated guide DNA and a series of complementary DNA targets undertaken in collaboration with the Yanli Wang laboratory (CAS Institute of Biophysics, Beijing, China). These ternary complex structures of cleavage-incompatible, cleavage-compatible and post-cleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a glutamate finger in generating a cleavage competent catalytic DEDD tetrad. Following cleavage, the seed segment forms a stable duplex, with the complementary segment of the target strand.

Sheng, G., Zhao, H., Wang, J., Rao, Y., Tian, W., Swarts, D. C, van der Oost, J., Patel, D. J. and Wang, Y. (2014). Structure-based cleavage mechanism of T. thermophiles Argonaute DNA guide strand-mediated DNA target cleavage. Proc. Natl. Acad. Scis. USA. 111, 652-657.

Nucleation, Propagation and Cleavage of Target RNAs in Eubacterial Ago Silencing Complexes

Nucleation, Propagation and Cleavage of Target RNAs in Eubacterial Ago Silencing Complexes

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here, we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5’-phosphprylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg2+-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3’-end of the guide from the PAZ pocket. Cleavage assays in the Thomas Tuschl laboratory (Rockefeller University) on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent ions observed in the crystal structures.

Wang, Y., Juranek, S., Li, H., Sheng, G., Wardle, G. S., Tuschl, T. & Patel, D. J. (2009). Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754-761.

Ternary Eubacterial Argonaute Silencing Complex Containing Guide-Target Duplex Spanning the Seed Segment

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 (Rockefeller University) 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 Eubacterial Argonaute Silencing Complex

Guide-Strand Containing Binary Eubacterial Argonaute Silencing Complex

The slicer activity of the RNA-induced silencing complex is associated with Argonaute, the RNase H-like PIWI domain of which catalyzes guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus Argonaute bound to a 5’-phosphorylated 21-base DNA guide stand, 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 of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies in the laboratory of our collaborator Thomas Tuschl (Rockefeller University) indicate that the key amino acid residues at the active site and those lining the 5’-phosphate-binding pocket made up of the MID domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3’-end-binding pocket made up of the PAZ domain 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.

Structure of a Eubacterial Argonaute with externally Bound siRNA

Argonaute Complex with Externally Bound siRNA

We have solved the crystal structures of Aquiflex aeolicus (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 (Rockefeller University), 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]

Eubacterial Argonautes are DNA Guide Strand-Mediated Site-Specific Endoribonucleases

Eubacterial Argonautes are DNA Guide Strand-Mediated Site-Specific Endoribonucleases

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies undertaken in the laboratory of our collaborator Thomas Tuschl (Rockefeller University), which establish this eubacterial Ago as a bonafide guide DNA strand-mediated site-specific RNA 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 RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent Ago2 reinforce the role of the PIWI domain in ‘slicer’ activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct 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.

piRNA Biogenesis

piRNAs serve as guides for transcriptional and posttranscriptional repression of diverse transposable elements in germ cells of metazoa. Current efforts are addressed at understanding the role of proteins and associated RNAs in mechanisms underlying piRNA biogenesis.

Recruitment of Aub and Ago3 to Nuage to form a Krimper assembled ping-pong complex

A project championed by the Alexei Aravin laboratory (Caltech) has demonstrated that Krimper interacts directly with Aubergine and Argonaute-3 to coordinate the assembly of the ping-pong piRNA processing complex.

Webster, A., Li, S., Hur, J. K., Wachsmuth, M., Bois, J., Perkins, E. M., Patel, D. J. and Aravin, A. A. (2015). Aub and Ago3 are recruited to nuage through two mechanisms to form a ping-pong complex assembled by Krimper. Mol. Cell 59, 564-575.

Transgenerationally inherited piRNAs trigger piRNA biogeneis

A project championed by the Alexei Aravin laboratory (Caltech) has demonstrated that transgenerationally inherited piRNAs act as an epigenetic memory for substrates of piRNA biogenesis at two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors.

Le Thomas, A., Stuwe, E., LI, S., Du, J.,,Marinov, G., Rozhkov, N., Chen, A. Y-C., Luo, Y., Sachidanandam, R., Tot, K. F., Patel, D. J. and Aravin, A.A. (2014). Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes Dev. 28, 1667-1680.

Recognition of siRNA and piRNA Ends by PAZ and MID-PIWI Domains

The PAZ and MID-PIWI domains are components of Argonaute and Piwi proteins. Structural studies have elucidated the role of the PAZ domain of Argonaute proteins in recognizing the 2’-OH ends of 3’-overhangs of siRNAs and the role of PAZ domain of Piwi proteins in recognizing the 2’-OCH3 modifications at the 3’-ends of piRNAs, as well as the role of the MID-PIWI domains of Argonaute proteins in targeting the 5’-phosphate of siRNAs.

Structural Basis for piRNA 2’-O-Methylated 3’-End Recognition by the PAZ Domain of Piwi Protein

Structural Basis for piRNA 2’-O-Methylated 3’-End Recognition by the PAZ Domain of Piwi Protein

Argonaute and Piwi proteins are key players in the RNA silencing pathway, with the former interacting with miRNAs and siRNAs, while the latter targets piRNAs that are 2’-O-methylated (2’-OCH3) at their 3’-ends. Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements, thereby protecting the genome against transposon-induced defects in gametogenesis and fertility. Humans contain four Piwi family proteins designated Hiwi1, Hiwi2, Hiwi3 and Hili. We report on the structures of Hili PAZ domain in the free state and Hiwi1 PAZ domain bound to self-complementary 14-mer RNAs (12-bp + 2-nt overhang) containing 2’-OCH3 and 2’-OH at their 3’-ends. These structures explain the molecular basis underlying accommodation of the 2’-OCH3 group within a preformed Hiwi1 PAZ domain binding pocket, whose hydrophobic characteristics account for the preferential binding of 2’-OCH3 over 2’-OH 3’-ends. These results contrast with the more restricted binding pocket for the human Ago1 PAZ domain, which exhibits a reverse order, with preferential binding of 2’-OH over 2’-OCH3 3’-ends.

Tian, Y., Simanshu, D., Ma, J. B. & Patel, D. J. (2011). Structural basis for piRNA 2’-O-methylated 3’-end recognition by the human Piwi PAZ domain. Proc. Natl. Acad. Scis. USA. 108, 903-910.

5’-Phosphate-Specific Recognition of siRNA by the MID-PIWI Domain

Encapsulation of siRNA 5'-Phosphate Ends by the Piwi Protein

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 (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 (Rockefeller University) 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]

2-nt 3’-Overhang-Specific Recognition of siRNA by the PAZ Domain

2-nt 3’-Overhang-Specific Recognition of siRNA by the PAZ Domain

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in some Argonaute and Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 Å crystal structure of the PAZ domain from human Argonaute eIFc1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3’-overhang of the siRNA duplex by binding the 7-nucleotide 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 siRNA-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.

Viral Suppressors of RNA Silencing

Plants use small interfering RNA (siRNAs), in a process called RNA silencing, to mediate sequence specific degradation of viral RNAs and thereby defend against attack. Viruses have organized a counter-defense whereby their genomes encode proteins pthat specifically suppress siRNA-mediated degradation.

Viral 2b Protein

In a collaborative study championed by the Nam-Hai Chua laboratory (Rockefeller University), it was demonstrated that that the Cucumber mosaic virus (CMV)-encoded 2b protein blocks AGO1 cleavage activity to inhibit miRNA pathways, attenuate RNA silencing and counter host defense.

Zhang, X., Yuan, Y-R., Pei, Y., Tuschl, T., Patel, D. J. & Chua, N-H. (2006). Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis AGO1 cleavage activity to counter plant defense. Genes Dev. 20, 3255-3268.

Viral p21 Protein

Viral p21 Protein

Many plant viruses encode proteins that suppress the antiviral RNA silencing response mounted by the host. The suppressors p19 from tombus virus and p21 from beet yellow virus appear to block silencing by directly binding siRNA, a critical mediator of the process. Here, we report the crystal structure of p21, which reveals an octameric ring architecture with a large central cavity of 90 Å diameter. The all α-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 is 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-binding protein, int/h2eracting with 21-nt or longer single- and double-stranded RNAs in vitro. This study reveals an RNA-binding structure adopted by the p21 silencing suppressor.

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.

Viral Tav2b Protein

Viral 2b Protein Sequesters siRNA Duplexes

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 Shou-Wei Ding laboratory (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.. Structural basis for siRNA recognition by a viral suppressor of non-cell autonomous RNA silencing.

Viral p19 Protein

Viral p19 Protein Measures siRNA Length

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 (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 ß-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 α-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]

RNA Tailing

RNAs undergo chemical modifications that can affect their activity, localization and stability. The current challenge has been to understand the molecular enzymatic machineries and biological functions of adenylation (A-tail) by Wispy and uridylation (U-tail) by TUTases at the 3’-end of RNA

Control of pre-miRNA fate by TUT7

A project championed by the Narry Kim laboratory (Seoul National University) has demonstrated how the TUTase TUT7 differentiates between pre-miRNA species with different overhangs thereby defining its role in the repair and removal of defective pre-miRNAs.

Kim, B., Ha, M., Leoff, L., Chang, H., Simanshu, D. K., Li, S., Patel, D. J., Joo, C. and Kim, V. N. (2015). TUT7 controls the fate of precursor miRNAs by using three different uridylation mechanisms. EMBO J. 34, 1801-1815.

Uridylation by TUT4 and TUT7 marks mRNA for degradation.

A project championed by the Narry Kim laboratory (Seoul National University, South Korea) identified TUT4 and TUT7 as mRNA uridylation enzymes, with uridylation readily occurring on deadenylated mRNAs in cells. These studies define the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.

Lim, J., Ha, M., Chang, H., Kwon, S. C., Simanshu, D. K., Patel, D. J. and Kim, V. N. (2015). Uridylation by TUT4 and TUT7 marks mRNA for degradation. Cell 159, 1365-1376.

Adenylation of maternally inherited miRNAs by Wispy

A project championed by the Narry Kim laboratory (Seoul National University, South Korea) have demonstrated that Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for adenylation of miRNAs at their 3’-ends in mature oocytes and early embryos in flies. These studies provide mechanistic insights into the regulation of maternal miRNAs and illustrate the importance of RNA tailing in development.

Lee, M., Choi, Y., Kim, K., Jin, H., Lim, J., Nguyen, T. A., Yang, J., Jeong, M., Giraldez, A. J., Yang, H., Patel, D. J. and Kim, V. N. (2015). Adenylation of maternally inherited microRNAs by Wispy. Mol Cell 56, 696-707.