The Structure of an RNAi Polymerase Links RNA Silencing and Transcription

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-Å resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.

[1]  E. V. Makeyev,et al.  A mechanism for initiating RNA-dependent RNA polymerization , 2001, Nature.

[2]  P. Cramer,et al.  Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution , 2001, Science.

[3]  E. Koonin,et al.  Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases , 2003, BMC Structural Biology.

[4]  G. Macino,et al.  Quelling in Neurospora crassa. , 2002, Advances in genetics.

[5]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[6]  D. Stuart,et al.  Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein. , 2005, Journal of structural biology.

[7]  E. Domingo,et al.  Structure of Foot-and-Mouth Disease Virus RNA-dependent RNA Polymerase and Its Complex with a Template-Primer RNA* , 2004, Journal of Biological Chemistry.

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

[9]  G. Macino,et al.  9 Quelling in Neurospora crassa , 2002 .

[10]  Kim Henrick,et al.  Detection of Protein Assemblies in Crystals , 2005, CompLife.

[11]  P. Cramer,et al.  Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution , 2001, Science.

[12]  S. Harrison,et al.  RNA Synthesis in a Cage—Structural Studies of Reovirus Polymerase λ3 , 2002, Cell.

[13]  K. Severinov,et al.  The Largest Subunits of RNA Polymerase from Gastric Helicobacters Are Tethered* , 1998, The Journal of Biological Chemistry.

[14]  D. Bamford,et al.  Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. , 2002, Molecular cell.

[15]  C. Mello,et al.  Revealing the world of RNA interference , 2004, Nature.

[16]  R. Martienssen,et al.  The role of RNA interference in heterochromatic silencing , 2004, Nature.

[17]  P. Willett,et al.  A graph-theoretic approach to the identification of three-dimensional patterns of amino acid side-chains in protein structures. , 1994, Journal of molecular biology.

[18]  Charles A. Lesburg,et al.  Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site , 1999, Nature Structural Biology.

[19]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[20]  G. Macino,et al.  Redundancy of the Two Dicer Genes in Transgene-Induced Posttranscriptional Gene Silencing in Neurospora crassa , 2004, Molecular and Cellular Biology.

[21]  Titia Sijen,et al.  On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing , 2001, Cell.

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

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

[24]  D. Stuart,et al.  What does structure tell us about virus evolution? , 2005, Current opinion in structural biology.

[25]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[26]  S. Gygi,et al.  Two RNAi Complexes, RITS and RDRC, Physically Interact and Localize to Noncoding Centromeric RNAs , 2004, Cell.

[27]  D I Stuart,et al.  Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 A. , 1979, Journal of molecular biology.

[28]  V S Lamzin,et al.  ARP/wARP and molecular replacement. , 2001, Acta crystallographica. Section D, Biological crystallography.

[29]  M. Rossmann,et al.  The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  H. L. Sänger,et al.  Isolation of an RNA-Directed RNA Polymerase–Specific cDNA Clone from Tomato , 1998, Plant Cell.

[31]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[32]  Russ Miller,et al.  The design and implementation of SnB version 2.0 , 1999 .

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

[34]  S. Yokoyama,et al.  Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution , 2002, Nature.

[35]  K. Severinov,et al.  Tethering of the Large Subunits of Escherichia coli RNA Polymerase* , 1997, The Journal of Biological Chemistry.

[36]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

[37]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[38]  N. Habuka,et al.  Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. , 1999, Structure.

[39]  M. Lai.  RNA Replication without RNA-Dependent RNA Polymerase: Surprises from Hepatitis Delta Virus , 2005, Journal of Virology.

[40]  G. Macino,et al.  Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase , 1999, Nature.

[41]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification , 2022 .

[42]  C Chothia,et al.  Surface, subunit interfaces and interior of oligomeric proteins. , 1988, Journal of molecular biology.

[43]  D. Bushnell,et al.  Structural Basis of Transcription Nucleotide Selection by Rotation in the RNA Polymerase II Active Center , 2004, Cell.

[44]  S. Harrison,et al.  RNA synthesis in a cage--structural studies of reovirus polymerase lambda3. , 2002, Cell.

[45]  Kenji Mizuguchi,et al.  A six-stranded double-psi β barrel is shared by several protein superfamilies , 1999 .