Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-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 four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, 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.     

Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex

RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes. Argonaute proteins, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5' end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5' nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5' nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3' overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute-siRNA guide complex.     

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.     

Structural basis for overhang-specific small interfering RNA recognition 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 Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 A crystal structure of the PAZ domain from human Argonaute eIF2c1 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-like duplex within a highly conserved binding pocket, and secures the 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.     

Structure of yeast Argonaute with guide RNA

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 ? crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, 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-bond 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.     

DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.     

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Box C/D RNA protein complexes (RNPs) direct site-specific 2'-O-methylation of RNA and ribosome assembly. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler. Despite many studies of C/D RNP structure, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-? resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide-substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.     

Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.     

Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides

Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.     

Structural basis of latency in plasminogen activator inhibitor-1.

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.     

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Helicases are molecular motors that use the energy of nucleoside 5'-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.     

Dicer recognizes the 5' end of RNA for efficient and accurate processing

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3' end but also the 5' end, with the cleavage site determined mainly by the distance (～22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in 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, 5'-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.     

Structural basis for broad DNA-specificity in integron recombination

Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. The lack of sequence conservation among attC sites has led to the hypothesis that a sequence-independent structural recognition determinant must exist within attC. Here we report the crystal structure of an integron integrase bound to an attC substrate. The structure shows that DNA target site recognition and high-order synaptic assembly are not dependent on canonical DNA but on the position of two flipped-out bases that interact in cis and in trans with the integrase. These extrahelical bases, one of which is required for recombination in vivo, originate from folding of the bottom strand of attC owing to its imperfect internal dyad symmetry. The mechanism reported here supports a new paradigm for how sequence-degenerate single-stranded genetic material is recognized and exchanged between bacteria.     

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.     

苦参凝集素的色氨酸残基修饰与荧光光谱研究

苦参凝集素（SFL）经等电聚焦测得其等电点为5.56,用比色法测得每分子SFL含有3个色氨酸残基,经N-溴代丁二酰亚胺（NBS）修饰表明其中2个Trp残基位于分子表面,当这2个Trp残基补修饰后,SFL的凝血活性完全丧失,糖保护试验表明,SFL专一性抑制糖Man能够保护SFL,阻断NBS对SFL的修饰作用,说明色氨酸不仅是SFL凝血活性所必需的,而且还参与其糖结合部位的组成,SFL随着NBS的逐渐修饰,其内源荧光光谱亦相应发生变化,结果表明分子表面的2个Trp残基可能位于分子的疏水袋中,而另一个Trp残基则埋藏于分子内部,荧光淬灭研究表明,丙烯酰胺能淬灭87%色氨酸残基的荧光。     

The human RNA kinase hClp1 is active on 3' transfer RNA exons and short interfering RNAs

RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer, synthetic siRNAs display a 5' OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5' end. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing and messenger RNA 3'-end formation machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5' end of the 3' exon during human tRNA splicing, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3'-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.     

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.