A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

[1]  U. Singh,et al.  RNAi Pathway Genes Are Resistant to Small RNA Mediated Gene Silencing in the Protozoan Parasite Entamoeba histolytica , 2014, PloS one.

[2]  D. Liu,et al.  RNA Isolation and Northern Blot Analysis , 2014 .

[3]  U. Singh,et al.  Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica , 2013, Nucleic acids research.

[4]  J. Doudna,et al.  Molecular mechanisms of RNA interference. , 2013, Annual review of biophysics.

[5]  N. Hall,et al.  Small RNA pyrosequencing in the protozoan parasite Entamoeba histolytica reveals strain-specific small RNAs that target virulence genes , 2013, BMC Genomics.

[6]  D. Bartel,et al.  Structure of yeast Argonaute with guide RNA , 2012, Nature.

[7]  Shane S. Sturrock,et al.  Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data , 2012, Bioinform..

[8]  U. Singh,et al.  Nucleus-localized Antisense Small RNAs with 5′-Polyphosphate Termini Regulate Long Term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain* , 2011, The Journal of Biological Chemistry.

[9]  R. Agami,et al.  MicroRNA regulation by RNA-binding proteins and its implications for cancer , 2011, Nature Reviews Cancer.

[10]  Makedonka Mitreva,et al.  Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles. , 2011, Genome research.

[11]  D. Bartel,et al.  The Inside-Out Mechanism of Dicers from Budding Yeasts , 2011, Cell.

[12]  E. Ullu,et al.  RNA Interference in Protozoan Parasites: Achievements and Challenges , 2011, Eukaryotic Cell.

[13]  Hyeshik Chang,et al.  Dicer recognizes the 5′ end of RNA for efficient and accurate processing , 2011, Nature.

[14]  Yi Liu,et al.  RNA Interference in Fungi: Pathways, Functions, and Applications , 2011, Eukaryotic Cell.

[15]  R. Ketting.  The many faces of RNAi. , 2011, Developmental cell.

[16]  Frederick P. Roth,et al.  Reconstitution of human RNA interference in budding yeast , 2011, Nucleic acids research.

[17]  U. Singh,et al.  RNA interference in Entamoeba histolytica: implications for parasite biology and gene silencing. , 2011, Future microbiology.

[18]  Yi Liu,et al.  Diverse small non-coding RNAs in RNA interference pathways. , 2011, Methods in molecular biology.

[19]  U. Singh,et al.  Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis , 2010, Eukaryotic Cell.

[20]  David W. Taylor,et al.  A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Catalytic Activity , 2010, Science.

[21]  G. Hannon,et al.  A dicer-independent miRNA biogenesis pathway that requires Ago catalysis , 2010, Nature.

[22]  Ayelet T. Lamm,et al.  Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma. , 2010, Molecular cell.

[23]  Pedro J. Batista,et al.  Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. , 2009, Molecular cell.

[24]  E. Ullu,et al.  Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei , 2009, Proceedings of the National Academy of Sciences.

[25]  Jeffrey P. Mower,et al.  RNAi in Budding Yeast , 2009, Science.

[26]  J. Hackney,et al.  Small RNAs with 5′-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica , 2008, PLoS pathogens.

[27]  R. Martienssen,et al.  RNAi, heterochromatin and the cell cycle. , 2008, Trends in genetics : TIG.

[28]  X. Ji.  The mechanism of RNase III action: how dicer dices. , 2008, Current topics in microbiology and immunology.

[29]  I. MacRae,et al.  Structural determinants of RNA recognition and cleavage by Dicer , 2007, Nature Structural &Molecular Biology.

[30]  U. Singh,et al.  Identification of an Entamoeba histolytica Serine-, Threonine-, and Isoleucine-Rich Protein with Roles in Adhesion and Cytotoxicity , 2007, Eukaryotic Cell.

[31]  D. Moazed,et al.  Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi. , 2007, Molecular cell.

[32]  Rashidul Haque,et al.  Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite , 2007, Cellular microbiology.

[33]  E. Astromskas,et al.  Tools and methods for genetic analysis of Saccharomyces castellii , 2007, Yeast.

[34]  Titia Sijen,et al.  Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class , 2007, Science.

[35]  Andrew Fire,et al.  Distinct Populations of Primary and Secondary Effectors During RNAi in C. elegans , 2007, Science.

[36]  E. Ullu,et al.  An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei. , 2006, RNA.

[37]  B. Bass,et al.  RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. , 2006, RNA.

[38]  V. Kim,et al.  The role of PACT in the RNA silencing pathway , 2006, The EMBO journal.

[39]  Angela N. Brooks,et al.  Structural Basis for Double-Stranded RNA Processing by Dicer , 2006, Science.

[40]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[41]  M. Abed,et al.  Molecular characterization of Entamoeba histolytica RNase III and AGO2, two RNA interference hallmark proteins. , 2005, Experimental parasitology.

[42]  A. Denli,et al.  Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein , 2005, PLoS biology.

[43]  Bernard B. Suh,et al.  The genome of the protist parasite Entamoeba histolytica , 2005, Nature.

[44]  D. Barford,et al.  Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity , 2004, The EMBO journal.

[45]  D. Court,et al.  Noncatalytic assembly of ribonuclease III with double-stranded RNA. , 2004, Structure.

[46]  Xiaodong Wang,et al.  R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway , 2003, Science.

[47]  V. Ambros,et al.  Role of MicroRNAs in Plant and Animal Development , 2003, Science.

[48]  B. Samuelsson,et al.  Ribonuclease activity and RNA binding of recombinant human Dicer , 2002, The EMBO journal.

[49]  C. Mello,et al.  The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1, and a DExH-Box Helicase to Direct RNAi in C. elegans , 2002, Cell.

[50]  D. Court,et al.  Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. , 2001, Structure.

[51]  A. Fire,et al.  Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans. , 2001, RNA.

[52]  Sherif Abou Elela,et al.  The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism. , 2001, Current issues in molecular biology.

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

[54]  E. Ullu,et al.  Accurate Transcription of the Trypanosoma brucei U2 Small Nuclear RNA Gene in a Homologous Extract (*) , 1995, The Journal of Biological Chemistry.

[55]  Tom Starzl,et al.  THE LANCET , 1992, The Lancet.

[56]  K. Köhrer,et al.  Preparation of high molecular weight RNA. , 1991, Methods in enzymology.

[57]  L S Diamond,et al.  A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. , 1978, Transactions of the Royal Society of Tropical Medicine and Hygiene.