Antisense RNA and RNAi in protozoan parasites: working hard or hardly working?

The complex life cycles of many protozoan parasites require the ability to respond to environmental and developmental cues through regulated gene expression. Traditionally, parasitologists have investigated these mechanisms by identifying and characterizing proteins that are necessary for the regulated expression of the genetic material. Although often successful, it is clear that protein-mediated gene regulation is only part of a complex story in which RNA itself is endowed with regulatory functions. Herein, we review both the known and potential regulatory roles of two types of RNA pathways within protozoan parasites: the RNA interference pathway and natural antisense transcripts. A better understanding of the native role of these pathways will not only enhance our understanding of the biology of these organisms but also aid in the development of more robust tools for reverse genetic analysis in this post-genomic era.

[1]  A. Riggs,et al.  The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. , 2005, RNA.

[2]  E. Ullu,et al.  Functional replacement of Trypanosoma brucei Argonaute by the human slicer Argonaute2. , 2006, RNA.

[3]  B. Kolli,et al.  Episomal Expression of Specific Sense and Antisense mRNAs in Leishmania amazonensis: Modulation of gp63 Level in Promastigotes and Their Infection of Macrophages In Vitro , 2000, Infection and Immunity.

[4]  S. Gottesman.  Micros for microbes: non-coding regulatory RNAs in bacteria. , 2005, Trends in genetics : TIG.

[5]  A. Bhattacharya,et al.  Blocking the expression of a calcium binding protein of the protozoan parasite Entamoeba histolytica by tetracycline regulatable antisense-RNA. , 2003, Molecular and biochemical parasitology.

[6]  Virander S. Chauhan,et al.  Double‐stranded RNA‐mediated gene silencing of cysteine proteases (falcipain‐1 and ‐2) of Plasmodium falciparum , 2002, Molecular microbiology.

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

[8]  E. Ullu,et al.  RNA interference in protozoan parasites , 2004, Cellular microbiology.

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

[10]  Brenda L. Bass,et al.  A developmentally regulated activity that unwinds RNA duplexes , 1987, Cell.

[11]  A. Djikeng,et al.  RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs. , 2001, RNA.

[12]  S. Ananvoranich,et al.  Single argonaute protein from Toxoplasma gondii is involved in the double-stranded RNA induced gene silencing. , 2006, International journal for parasitology.

[13]  T. Wellems,et al.  Malaria: Cooperative silencing elements in var genes , 2001, Nature.

[14]  E. Ullu,et al.  Selection and Characterization of RNA Interference-Deficient Trypanosomes Impaired in Target mRNA Degradation , 2004, Eukaryotic Cell.

[15]  S. Parmley,et al.  The Expression of Lactate Dehydrogenase Is Important for the Cell Cycle of Toxoplasma gondii* , 2004, Journal of Biological Chemistry.

[16]  J. Donelson,et al.  Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi. , 2004, Molecular and biochemical parasitology.

[17]  M. C. Touz,et al.  A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia , 2005, Molecular microbiology.

[18]  K. Gull,et al.  Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Virander S. Chauhan,et al.  In vivo gene silencing in Plasmodium berghei--a mouse malaria model. , 2003, Biochemical and biophysical research communications.

[20]  E. Ullu,et al.  Small Sense and Antisense RNAs Derived from a Telomeric Retroposon Family in Giardia intestinalis , 2005, Eukaryotic Cell.

[21]  C. Wang,et al.  The double-stranded RNA in Trichomonas vaginalis may originate from virus-like particles. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Shoshi Kikuchi,et al.  Antisense transcripts with rice full-length cDNAs , 2003, Genome Biology.

[23]  J. Boothroyd,et al.  Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short, identical exon at their 5' end. , 1982, Gene.

[24]  S. Gallati,et al.  Overlapping sense and antisense transcription units in Trypanosoma brucei , 2001, Molecular microbiology.

[25]  J. Donelson,et al.  Tetracycline-regulated RNA interference in Trypanosoma congolense. , 2002, Molecular and biochemical parasitology.

[26]  E. Ullu,et al.  Expression site silencing and life-cycle progression appear normal in Argonaute1-deficient Trypanosoma brucei. , 2006, Molecular and biochemical parasitology.

[27]  M. Matzke,et al.  Transcriptional silencing and promoter methylation triggered by double‐stranded RNA , 2000, The EMBO journal.

[28]  Catherine Vaquero,et al.  PfMyb1, a Plasmodium falciparum transcription factor, is required for intra-erythrocytic growth and controls key genes for cell cycle regulation. , 2005, Journal of molecular biology.

[29]  P. Myler,et al.  Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major. , 2003, International journal for parasitology.

[30]  G. Fink,et al.  Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae , 2006, Cell.

[31]  M. Bienz,et al.  Quantitative assessment of sense and antisense transcripts from genes involved in antigenic variation (vsp genes) and encystation (cwp 1 gene) of Giardia lamblia clone GS/M-83-H7 , 2004, Parasitology.

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

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

[34]  S. Beverley,et al.  Characterization of quinonoid-Dihydropteridine Reductase (QDPR) from the Lower Eukaryote Leishmania major * , 2002, The Journal of Biological Chemistry.

[35]  Yitzhak Pilpel,et al.  Genome‐wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms , 2006, EMBO reports.

[36]  Erez Y. Levanon,et al.  Widespread occurrence of antisense transcription in the human genome , 2003, Nature Biotechnology.

[37]  S. Beverley,et al.  Transcriptional mapping of the amplified region encoding the dihydrofolate reductase-thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs , 1989, Molecular and cellular biology.

[38]  C. Wang,et al.  Discovery of a specific double-stranded RNA virus in Giardia lamblia. , 1986, Molecular and biochemical parasitology.

[39]  Marie-Agnès Dillies,et al.  Transcriptome analysis of antigenic variation in Plasmodium falciparum - var silencing is not dependent on antisense RNA , 2005, Genome Biology.

[40]  N. Yang,et al.  L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells , 2006, Nature Structural &Molecular Biology.

[41]  G. McConkey,et al.  Analysis of short RNAs in the malaria parasite and its red blood cell host , 2006, FEBS letters.

[42]  O. White,et al.  Analysis of the transcriptome of the protozoan Theileria parva using MPSS reveals that the majority of genes are transcriptionally active in the schizont stage , 2005, Nucleic acids research.

[43]  T. Hall,et al.  Structure and function of argonaute proteins. , 2005, Structure.

[44]  S. Ananvoranich,et al.  Double-stranded RNA can mediate the suppression of uracil phosphoribosyltransferase expression in Toxoplasma gondii. , 2003, Biochemical and biophysical research communications.

[45]  E. Rapaport,et al.  Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[46]  T. Nash,et al.  The abundance of sterile transcripts in Giardia lamblia. , 2001, Nucleic acids research.

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

[48]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[49]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[50]  H. Elmendorf,et al.  Bidirectional transcription is an inherent feature of Giardia lamblia promoters and contributes to an abundance of sterile antisense transcripts throughout the genome , 2007, Nucleic acids research.

[51]  S. Ananvoranich,et al.  Comparative analysis of antisense RNA, double-stranded RNA, and delta ribozyme-mediated gene regulation in Toxoplasma gondii. , 2002, Antisense & nucleic acid drug development.

[52]  A. Kucknoor,et al.  Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis , 2007, BMC Microbiology.

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

[54]  E. Birney,et al.  Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs , 2002, Nature.

[55]  F. Gillin,et al.  Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. , 2007, Molecular and biochemical parasitology.

[56]  D. Wirth,et al.  RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum. , 2005, RNA.

[57]  Jay Shendure,et al.  Computational discovery of sense-antisense transcription in the human and mouse genomes , 2002, Genome Biology.

[58]  D. Holt,et al.  Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA. , 2000, Molecular and biochemical parasitology.

[59]  D. Klumpp,et al.  Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis , 2004, Molecular microbiology.

[60]  C. Wanidworanun,et al.  Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum. , 1999, Molecular and biochemical parasitology.

[61]  P. Bastin,et al.  TbAGO1, an Argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei , 2003, BMC Biology.

[62]  J. Kawai,et al.  Collection, Mapping, and Annotation of Over 28,000 cDNA Clones from japonica Rice , 2003, Science.

[63]  E. Wagner,et al.  Imprinted expression of the Igf2r gene depends on an intronic CpG island , 1997, Nature.

[64]  Jonathan Schug,et al.  Widespread distribution of antisense transcripts in the Plasmodium falciparum genome. , 2004, Molecular and biochemical parasitology.

[65]  B. Haas,et al.  The Genome Sequence of Trypanosoma cruzi, Etiologic Agent of Chagas Disease , 2005, Science.

[66]  L. M. Cummings,et al.  Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite. , 2001, Molecular biology of the cell.

[67]  C. Dumas,et al.  A Novel Class of Developmentally Regulated Noncoding RNAs in Leishmania , 2006, Eukaryotic Cell.

[68]  G. McConkey,et al.  RNA interference (RNAi) inhibits growth of Plasmodium falciparum. , 2002, Molecular and biochemical parasitology.

[69]  Ji-Joon Song,et al.  Purified Argonaute2 and an siRNA form recombinant human RISC , 2005, Nature Structural &Molecular Biology.

[70]  Brian White,et al.  Comparative genomic analysis of three Leishmania species that cause diverse human disease , 2007, Nature Genetics.

[71]  S. Beverley,et al.  Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania. , 2003, Molecular and biochemical parasitology.

[72]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[73]  C. Weber,et al.  Double-stranded RNA mediates homology-dependent gene silencing of gamma-tubulin in the human parasite Entamoeba histolytica. , 2004, Molecular and biochemical parasitology.

[74]  P. Das,et al.  The 29-Kilodalton Thiol-Dependent Peroxidase of Entamoeba histolytica Is a Factor Involved in Pathogenesis and Survival of the Parasite during Oxidative Stress , 2007, Eukaryotic Cell.

[75]  S. Ananvoranich,et al.  RNA Silencing of Glycolysis Pathway in Toxoplasma gondii , 2006, The Journal of eukaryotic microbiology.

[76]  M. Blackman.  RNAi in protozoan parasites: what hope for the Apicomplexa? , 2003, Protist.

[77]  H. Cerutti,et al.  On the origin and functions of RNA-mediated silencing: from protists to man , 2006, Current Genetics.

[78]  E. Ullu,et al.  Function of the Trypanosome Argonaute 1 Protein in RNA Interference Requires the N-terminal RGG Domain and Arginine 735 in the Piwi Domain* , 2004, Journal of Biological Chemistry.

[79]  R. Benne,et al.  Major transcript of the frameshifted coxll gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA , 1986, Cell.

[80]  Fangli Lu,et al.  cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome , 2007, BMC Genomics.

[81]  B. Wickstead,et al.  Repetitive Elements in Genomes of Parasitic Protozoa , 2003, Microbiology and Molecular Biology Reviews.

[82]  G. Matlashewski,et al.  Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[83]  A. Djikeng,et al.  Argonaute Protein in the Early Divergent Eukaryote Trypanosoma brucei: Control of Small Interfering RNA Accumulation and Retroposon Transcript Abundance , 2004, Molecular and Cellular Biology.

[84]  L. Gedamu,et al.  Functional analysis of cathepsin B-like cysteine proteases from Leishmania donovani complex. Evidence for the activation of latent transforming growth factor beta. , 2002, The Journal of biological chemistry.

[85]  G. Matlashewski,et al.  Analysis of antisense and double stranded RNA downregulation of A2 protein expression in Leishmania donovani. , 2000, Molecular and biochemical parasitology.

[86]  R. Ramaswamy,et al.  The LINEs and SINEs of Entamoeba histolytica: comparative analysis and genomic distribution. , 2005, Experimental parasitology.

[87]  S. Kyes,et al.  Stage-specific merozoite surface protein 2 antisense transcripts in Plasmodium falciparum. , 2002, Molecular and biochemical parasitology.

[88]  X. Su,et al.  The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of plasmodium falciparum-infected erythrocytes , 1995, Cell.

[89]  F. Gillin,et al.  Developmental gene regulation in Giardia lamblia: first evidence for an encystation‐specific promoter and differential 5′ mRNA processing , 1999, Molecular microbiology.

[90]  S. Barik,et al.  Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference , 2002, Malaria Journal.

[91]  Shulamit Michaeli,et al.  Small nucleolar RNA interference induced by antisense or double-stranded RNA in trypanosomatids , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[92]  Aaron J Mackey,et al.  The transcriptome of Toxoplasma gondii , 2005, BMC Biology.

[93]  A. Lohia,et al.  Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica. , 2004, Biochemical and biophysical research communications.

[94]  V. Gvozdev,et al.  Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline , 2005, Nucleic acids research.

[95]  H. Margalit,et al.  A survey of small RNA-encoding genes in Escherichia coli. , 2003, Nucleic acids research.

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

[97]  D. Mirelman,et al.  Inhibition of gene expression in Entamoeba by the transcription of antisense RNA: effect of 5' and 3' regulatory elements. , 2000, Molecular and biochemical parasitology.

[98]  L. Timmons.  Endogenous inhibitors of RNA interference in Caenorhabditis elegans. , 2004, BioEssays : news and reviews in molecular, cellular and developmental biology.

[99]  A. Djikeng,et al.  An siRNA ribonucleoprotein is found associated with polyribosomes in Trypanosoma brucei. , 2003, RNA.

[100]  R. Barker,et al.  Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[101]  Richard D. Hayes,et al.  Draft Genome Sequence of the Sexually Transmitted Pathogen Trichomonas vaginalis , 2007, Science.

[102]  G. Barber,et al.  The dsRNA binding protein family: critical roles, diverse cellular functions , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[103]  T. Wellems,et al.  Plasmodium falciparum var Genes Are Regulated by Two Regions with Separate Promoters, One Upstream of the Coding Region and a Second within the Intron* , 2003, Journal of Biological Chemistry.