HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing

We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the ‘dicer-related-helicase’ from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF− cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF− cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways.

[1]  Frank Lyko,et al.  Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi , 2005, Nucleic acids research.

[2]  Edwards Allen,et al.  DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  David L. Steffen,et al.  The genome of the social amoeba Dictyostelium discoideum , 2005, Nature.

[4]  Olivier Voinnet,et al.  Induction and suppression of RNA silencing: insights from viral infections , 2005, Nature Reviews Genetics.

[5]  S. Gräf,et al.  A database search for double-strand containing RNAs in Dictyostelium discoideum , 2004, Biological chemistry.

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

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

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

[9]  Olivier Voinnet,et al.  RETRACTED: Probing the MicroRNA and Small Interfering RNA Pathways with Virus-Encoded Suppressors of RNA Silencing[W] , 2004, Plant Cell.

[10]  Gary Ruvkun,et al.  A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans , 2004, Nature.

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

[12]  Kazuo Shinozaki,et al.  Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in Arabidopsis thaliana , 2004, Plant Molecular Biology.

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

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

[15]  A. Depicker,et al.  RNA Target Sequences Promote Spreading of RNA Silencing1 , 2003, Plant Physiology.

[16]  Sandhya P Koushika,et al.  Loss of the Putative RNA-Directed RNA Polymerase RRF-3 Makes C. elegans Hypersensitive to RNAi , 2002, Current Biology.

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

[18]  Matthias Platzer,et al.  Sequence and analysis of chromosome 2 of Dictyostelium discoideum , 2002, Nature.

[19]  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.

[20]  T. Steck,et al.  RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase. , 2002, Molecular biology of the cell.

[21]  W. Filipowicz,et al.  Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines , 2001, Proceedings of the National Academy of Sciences of the United States of America.

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

[23]  A. Fire,et al.  Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. , 2001, Gene.

[24]  S. Diegel,et al.  Dictyostelium double-stranded ribonuclease. , 2001, Methods in enzymology.

[25]  J. Spudich,et al.  Dynacortin, a Genetic Link between Equatorial Contractility and Global Shape Control Discovered by Library Complementation of a Dictyostelium discoideum Cytokinesis Mutant , 2000, The Journal of cell biology.

[26]  Tamas Dalmay,et al.  An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus , 2000, Cell.

[27]  J. Spoerke,et al.  EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans , 2000, Current Biology.

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

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

[30]  T. Hubbard,et al.  Using neural networks for prediction of the subcellular location of proteins. , 1998, Nucleic acids research.

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

[32]  F. Půta,et al.  Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivation of genes in Dictyostelium. , 1998, Folia biologica.

[33]  G. Cavet,et al.  On the role of myosin-II in cytokinesis: division of Dictyostelium cells under adhesive and nonadhesive conditions. , 1997, Molecular biology of the cell.

[34]  M. Maniak,et al.  Developmental regulation of antisense-mediated gene silencing in Dictyostelium. , 1994, Antisense research and development.

[35]  P. Morandini,et al.  Mutants of Dictyostelium discoideum with defects in the regulation of discoidin I expression. , 1993, Developmental biology.

[36]  K. Sutoh.  A transformation vector for dictyostelium discoideum with a new selectable marker bsr. , 1993, Plasmid.

[37]  D. St Johnston,et al.  A conserved double-stranded RNA-binding domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[38]  P Linder,et al.  D‐E‐A‐D protein family of putative RNA helicases , 1992, Molecular microbiology.

[39]  G. Gerisch,et al.  Screening for Dictyostelium mutants defective in cytoskeletal proteins by colony immunoblotting. , 1991, Methods in enzymology.

[40]  P. Slonimski,et al.  Birth of the D-E-A-D box , 1989, Nature.

[41]  M. Maniak,et al.  A colony-blot technique for the detection of specific transcripts in eukaryotes. , 1989, Analytical biochemistry.

[42]  A. Noegel,et al.  Homologous recombination in the Dictyostelium alpha‐actinin gene leads to an altered mRNA and lack of the protein. , 1987, The EMBO journal.

[43]  R. Firtel,et al.  Chapter 4 Molecular Biology in Dictyostelium: Tools and Applications , 1987 .

[44]  R. Firtel,et al.  Molecular biology in Dictyostelium: tools and applications. , 1987, Methods in cell biology.

[45]  R. Gomer,et al.  Phenocopy of discoidin I-minus mutants by antisense transformation in Dictyostelium , 1985, Cell.

[46]  R. Firtel,et al.  High-copy-number transformants and co-transformation in Dictyostelium. , 1985, Gene.