THE INTERNATIONAL JOURNAL OF Int J Dev Biol 53 245 257 2009 DEVELOPMENTAL BIOLOGY doi 10 1387 ijdb 082691av www intjdevbiol com Common themes in siRNA mediated epigenetic silencing pathways ANDR VERDEL AUR LIA VAVASSEUR MADALEN LE GORREC and LEILA TOUAT TODESCHINI Institut National de la Sant et de la Recherche M dicale INSERM U823 and Universit Joseph Fourier Institut Albert Bonniot Facult de M decine Grenoble France ABSTRACT Small interfering RNAs siRNAs act through RNA interference RNAi pathways to silence gene expression either at the transcriptional or post transcriptional level Here we review mechanisms and functions of siRNA mediated silencing pathways that promote chromatin modifications in the fission yeast Schizosaccharomyces pombe plants and animals In fission yeast siRNAs are involved in heterochromatin formation and key aspects of the underlying siRNAdependent pathway have been uncovered Two RNAi complexes the RNA Induced Transcriptional Silencing complex RITS which contains a siRNA bound to an Argonaute protein and the RNA Directed RNA polymerase Complex RDRC are critical components of the pathway In addition this pathway implicates non coding nascent transcripts synthesized by RNA polymerase II RNApII and the RNApII itself In Arabidopsis thaliana the RNA directed DNA methylation RdDM pathway appears to share a similar set of proteins and enzymatic activities suggesting that beyond certain aspects that are specific to each pathway part of the siRNA mediated epigenetic silencing mechanisms are conserved between fission yeast and plants Moreover in both organisms the pathways target repetitive DNA sequences This conservation of mechanisms and genomic targets might actually extend to animals as recent investigations revealed the existence of endogenous siRNA based pathways directed against repetitive DNA sequences in flies and mammals KEY WORDS siRNA RNAi gene silencing epigenetics heterochromatin Introduction Just over a decade ago Fire Mello and colleagues discovered that micro injection of long double stranded RNA dsRNA into Caenhorbditis elegans worms triggered a potent and sequencespecific gene silencing or RNA interference RNAi Fire et al 1998 Mello and Conte 2004 It was rapidly found that small RNA molecules of 21 to 25 nucleotides termed small interfering RNAs siRNAs are the molecules that convey RNAi sequence specificity Mello and Conte 2004 SiRNA mediated silencing has now been observed in the vast majority of the eukaryotes studied with the noticeable exception of the yeast Saccharomyces cerevisiae is widely used to analyze the function of proteins by knocking down their expression and offers the promise of new therapeutic strategies de Fougerolles et al 2007 Mello and Conte 2004 Besides siRNAs two other major species of small RNAs the micro RNAs miRNAs and the Piwi associated RNAs piRNAs have been shown to also act through RNAi or RNAi related pathways These three small RNA species can be distinguished based on their biogenesis and length the identity of their Argonaute partner and their associated mechanism of gene silencing Argonaute proteins form a superfamily composed of the Argonaute and the Piwi clades MiRNAs like siRNAs are generated from double stranded RNA through a dicing catalyzed by the Dicer nuclease are of similar size and bind members of the Argonaute clade However unlike siRNAs miRNAs do not originate from double stranded RNAs formed by the hybridization of two complementary RNAs MiRNAs are generated from RNA molecules that individually form small hairpin RNA structures of 70 nucleotides Abbreviations used in this paper RDRC RNA directed RNA polymerase complex RdDM RNA directed DNA methylation RITS RNA induced transcriptional silencing complex RNAi RNA interference RNApII RNA polymerase II siRNA small interfering RNA TGS transcriptional gene silencing Address correspondence to Dr Andr Verdel Albert Bonniot Institute INSERM UJF Research Center U823 Department of Cell Differentiation and Transformation Domaine de la Merci 38706 La Tronche Cedex France Fax 33 4 7554 9595 e mail andre verdel ujf grenoble fr web http www iab ujf grenoble fr Published online 21 April 2009 ISSN Online 1696 3547 Print 0214 6282 2009 UBC Press Printed in Spain 246 A Verdel et al Fig 1 A Post transcriptional and B tran A B scriptional siRNA mediated silencing pathways Both pathways are activated by the detection of long dsRNAs by the RNAi machinery DsRNAs are processed by Dicer ribonucleases Dcr into siRNA duplexes that then bind to Argonaute Ago proteins Together with the siRNA Ago proteins form the core of RNAi effector complexes RISC and RITS One strand of the incorporated doublestranded siRNA is eliminated the passenger strand whereas the other strand the guide strand is used to recognize the target RNA Perfect base pairing between the guide strand and the target RNA allows the cleavage of the target Thus both PTGS and TGS pathways share a common set of mechanisms and enzymatic activities highlighted by the central large arrow Nonetheless the silencing induced by both pathways shows major distinctions In the post transcriptional gene silencing PTGS siRNAs direct mRNA degradation in the cytoplasm with no epigenetic incidence as they do not act directly on the transcription rate of the corresponding gene On the contrary siRNA mediated transcriptional gene silencing TGS acts in the nucleus and comes with chromatin modifications that silence transcription and that can be maintained throughout mitosis and meiosis thus maintaining silencing over multiple generations Note that the PTGS pathway is illustrated with the generic names of protein and complexes whereas for the TGS pathway the fission yeast names are mentioned RISC RNA Induced Silencing Complex RITS RNA Induced Transcriptional Silencing complex Grey circles represent nucleosomes Bartel 2004 Once loaded onto RNAi effector complexes the miRNA sequence allows the RNAi complex to base pair with target mRNAs but unlike siRNAs the miRNA sequence often presents several mismatches with its target Moreover for most of the organisms studied the miRNA induces a silencing at the posttranscriptional level in the cytoplasm that consists in inhibiting translation whereas siRNAs can act in the nucleus directly on the gene PiRNAs have been discovered more recently Aravin et al 2007 Their generation does not depend on Dicer they have a longer average size of 28 to 33 nucleotides and conversely to siRNAs and miRNAs that bind