Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21–25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar‐binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C‐terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H‐like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H‐like domain may possess nuclease activity. The conserved region around the C‐terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5′ phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA.

[1]  V. Ambros,et al.  The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. , 1999, Developmental biology.

[2]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

[3]  T. Tuschl,et al.  Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. , 2004, Molecular cell.

[4]  G. Szittya,et al.  Size Selective Recognition of siRNA by an RNA Silencing Suppressor , 2003, Cell.

[5]  Martin Tabler,et al.  Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila , 2001, Current Biology.

[6]  G. Ruvkun,et al.  Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans , 1993, Cell.

[7]  T. Steitz,et al.  Recombining the structures of HIV integrase, RuvC and RNase H. , 1995, Structure.

[8]  D. Bartel,et al.  MicroRNA-Directed Cleavage of HOXB8 mRNA , 2004, Science.

[9]  S. Ding,et al.  Induction and Suppression of RNA Silencing by an Animal Virus , 2002, Science.

[10]  Andrew Fire,et al.  The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans , 1999, Cell.

[11]  Phillip D Zamore,et al.  The RNA-Induced Silencing Complex Is a Mg2+-Dependent Endonuclease , 2004, Current Biology.

[12]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

[13]  Michael Q. Zhang,et al.  The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. , 2002, Genes & development.

[14]  B. Simon,et al.  Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain , 2003, Nature.

[15]  Xiaofeng Cao,et al.  ARGONAUTE4 Control of Locus-Specific siRNA Accumulation and DNA and Histone Methylation , 2003, Science.

[16]  Akira Ishizuka,et al.  Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. , 2004, Genes & development.

[17]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

[18]  N. Rhind,et al.  A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. , 2004, Genes & development.

[19]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

[20]  Henning Urlaub,et al.  Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi , 2002, Cell.

[21]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[22]  Phillip D Zamore,et al.  Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. , 2002, Molecular cell.

[23]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[24]  G. Hannon,et al.  Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity , 2004, Science.

[25]  Javier F. Palatnik,et al.  Control of leaf morphogenesis by microRNAs , 2003, Nature.

[26]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.

[27]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[28]  B. Bass,et al.  A Role for the RNase III Enzyme DCR-1 in RNA Interference and Germ Line Development in Caenorhabditis elegans , 2001, Science.

[29]  V. Kim,et al.  MicroRNA maturation: stepwise processing and subcellular localization , 2002, The EMBO journal.

[30]  S. Jayasena,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[31]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[32]  S. Kim,et al.  Crystal structure of archaeal RNase HII: a homologue of human major RNase H. , 2000, Structure.

[33]  A. Pasquinelli,et al.  A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA , 2001, Science.

[34]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[35]  P. Zamore,et al.  Kinetic analysis of the RNAi enzyme complex , 2004, Nature Structural &Molecular Biology.

[36]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[37]  C. Llave,et al.  Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA , 2002, Science.

[38]  M. Montgomery.  RNA interference: historical overview and significance. , 2004, Methods in molecular biology.

[39]  James A. Birchler,et al.  Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery , 2004, Science.

[40]  D. Patel,et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain , 2004, Nature.

[41]  Serge Batalov,et al.  Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. , 2003, Molecular cell.

[42]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[43]  E. Sontheimer,et al.  Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways , 2004, Cell.

[44]  K. Morris,et al.  Small Interfering RNA-Induced Transcriptional Gene Silencing in Human Cells , 2004, Science.

[45]  W. Filipowicz,et al.  Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer , 2004, EMBO reports.

[46]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[47]  Ira M. Hall,et al.  Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi , 2002, Science.

[48]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

[49]  D. Baulcombe.  RNA silencing in plants , 2004, Nature.

[50]  Ira M. Hall,et al.  RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Ji-Joon Song,et al.  The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes , 2003, Nature Structural Biology.

[52]  T. Rana,et al.  siRNA function in RNAi: a chemical modification analysis. , 2003, RNA.

[53]  G. Hannon,et al.  C . elegans involved in developmental timing in Dicer functions in RNA interference and in synthesis of small RNA , 2001 .

[54]  Thomas Tuschl,et al.  RISC is a 5' phosphomonoester-producing RNA endonuclease. , 2004, Genes & development.

[55]  Ronald H. A. Plasterk,et al.  Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi , 2003, Nature.

[56]  Phillip A. Sharp,et al.  The RNAi revolution , 2004, Nature.

[57]  Nick V Grishin,et al.  Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[58]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[59]  A. Caudy,et al.  A micrococcal nuclease homologue in RNAi effector complexes , 2003, Nature.

[60]  E. Sontheimer,et al.  A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila , 2004, Cell.

[61]  G. Hutvagner,et al.  A microRNA in a Multiple-Turnover RNAi Enzyme Complex , 2002, Science.

[62]  B. Samuelsson,et al.  Dicer is required for chromosome segregation and gene silencing in fission yeast cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[63]  L. Malinina,et al.  Recognition of small interfering RNA by a viral suppressor of RNA silencing , 2003, Nature.

[64]  A. Caudy,et al.  Fragile X-related protein and VIG associate with the RNA interference machinery. , 2002, Genes & development.

[65]  Songtao Jia,et al.  RNAi-Mediated Targeting of Heterochromatin by the RITS Complex , 2004, Science.

[66]  T. Rana,et al.  RNAi in human cells: basic structural and functional features of small interfering RNA. , 2002, Molecular cell.

[67]  Michael Sattler,et al.  Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain , 2004, Nature Structural &Molecular Biology.

[68]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

[69]  M. Oshimura,et al.  Dicer is essential for formation of the heterochromatin structure in vertebrate cells , 2004, Nature Cell Biology.

[70]  Hajime Sakai,et al.  Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016238. , 2003, The Plant Cell Online.

[71]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature Reviews Genetics.

[72]  T. Du,et al.  RISC Assembly Defects in the Drosophila RNAi Mutant armitage , 2004, Cell.

[73]  W. Wooster,et al.  Crystal structure of , 2005 .