Tethering RITS to a Nascent Transcript Initiates RNAi- and Heterochromatin-Dependent Gene Silencing

In the fission yeast Schizosaccharomyces pombe, the RNA-Induced Transcriptional Silencing (RITS) complex has been proposed to target the chromosome via siRNA-dependent base-pairing interactions to initiate heterochromatin formation. Here we show that tethering of the RITS subunit, Tas3, to the RNA transcript of the normally active ura4+ gene silences ura4+ expression. This silencing depends on a functional RNAi pathway, requires the heterochromatin proteins, Swi6/HP1, Clr4/Suv39h, and Sir2, and is accompanied by the generation of ura4+ siRNAs, histone H3-lysine 9 methylation, and Swi6 binding. Furthermore, the ability of the newly generated ura4+ siRNAs to silence a second ura4+ allele in trans is strongly inhibited by the conserved siRNA nuclease, Eri1. Surprisingly, silencing of tethered ura4+, or ura4+ inserted within centromeric heterochromatin, or some of the endogenous centromeric repeat promoters, is not associated with changes in RNA polymerase II occupancy. These findings support a model in which targeting of nascent transcripts by RITS mediates chromatin modifications and suggest that cotranscriptional processing events play a primary role in the silencing mechanism.

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

[2]  G. Hannon,et al.  Unlocking the potential of the human genome with RNA interference , 2004, Nature.

[3]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[4]  R. Pillai.  MicroRNA function: multiple mechanisms for a tiny RNA? , 2005, RNA.

[5]  D. Moazed,et al.  Common themes in mechanisms of gene silencing. , 2001, Molecular cell.

[6]  D. Moazed,et al.  Sir2 Regulates Histone H3 Lysine 9 Methylation and Heterochromatin Assembly in Fission Yeast , 2003, Current Biology.

[7]  S. Grewal.  Transcriptional silencing in fission yeast , 2000, Journal of cellular physiology.

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

[9]  R. Plasterk,et al.  The silence of the genes. , 2000, Current opinion in genetics & development.

[10]  M. Hentze,et al.  Using the lambdaN peptide to tether proteins to RNAs. , 2004, Methods in molecular biology.

[11]  R. Allshire,et al.  Distinct protein interaction domains and protein spreading in a complex centromere. , 2000, Genes & development.

[12]  A. Hoffmann,et al.  Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe , 1989, Molecular and cellular biology.

[13]  Brian D. Strahl,et al.  Role of Histone H3 Lysine 9 Methylation in Epigenetic Control of Heterochromatin Assembly , 2001, Science.

[14]  Michael T. McManus,et al.  Gene silencing in mammals by small interfering RNAs , 2002, Nature Reviews Genetics.

[15]  T. Sugiyama,et al.  RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing , 2004, Nature Genetics.

[16]  R. Martienssen,et al.  RNA Polymerase II Is Required for RNAi-Dependent Heterochromatin Assembly , 2005, Science.

[17]  Y. Schwartz,et al.  Polycomb silencing blocks transcription initiation. , 2004, Molecular cell.

[18]  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.

[19]  T. Sugiyama,et al.  RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Moazed,et al.  Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing. , 2003, Genes & development.

[21]  D. Moazed,et al.  A Cullin E3 Ubiquitin Ligase Complex Associates with Rik1 and the Clr4 Histone H3-K9 Methyltransferase and is Required for RNAi-Mediated Heterochromatin Formation , 2005, RNA biology.

[22]  N. Rhind,et al.  A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. , 2004, Genes & development.

[23]  V. Orlando,et al.  General transcription factors bind promoters repressed by Polycomb group proteins , 2001, Nature.

[24]  Ira M. Hall,et al.  Establishment and Maintenance of a Heterochromatin Domain , 2002, Science.

[25]  D. Moazed,et al.  RNAi‐directed assembly of heterochromatin in fission yeast , 2005, FEBS letters.

[26]  R. Allshire,et al.  Position effect variegation at fission yeast centromeres , 1994, Cell.

[27]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[28]  T. Tuschl,et al.  Mechanisms of gene silencing by double-stranded RNA , 2004, Nature.

[29]  A. Caudy,et al.  A micrococcal nuclease homologue in RNAi effector complexes , 2003, Nature.

[30]  M. Hentze,et al.  Using the λN Peptide to Tether Proteins to RNAs , 2004 .

[31]  James A. Birchler,et al.  Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery , 2004, Science.

[32]  V. Pirrotta,et al.  Epigenetic silencing mechanisms in budding yeast and fruit fly: different paths, same destinations. , 2005, Molecular cell.

[33]  G. Hannon.  RNA interference : RNA , 2002 .

[34]  Phillip D Zamore,et al.  Ancient Pathways Programmed by Small RNAs , 2002, Science.

[35]  S. Elgin,et al.  Epigenetic Codes for Heterochromatin Formation and Silencing Rounding up the Usual Suspects , 2002, Cell.

[36]  P. Philippsen,et al.  Heterologous modules for efficient and versatile PCR‐based gene targeting in Schizosaccharomyces pombe , 1998, Yeast.

[37]  Gregory J. Hannon,et al.  Insight Review Articles , 2022 .

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

[39]  S. Peltz,et al.  The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. , 1991, Genes & development.

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

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

[42]  K. Morris,et al.  Small Interfering RNA-Induced Transcriptional Gene Silencing in Human Cells , 2004, Science.

[43]  P. Jeffrey,et al.  Regulation of Heterochromatic Silencing and Histone H 3 Lysine-9 Methylation by RNAi , 2002 .

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

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

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

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

[48]  M. Hentze,et al.  Y14 and hUpf3b form an NMD-activating complex. , 2003, Molecular cell.

[49]  Sean D. Taverna,et al.  Methylation of Histone H3 at Lysine 9 Targets Programmed DNA Elimination in Tetrahymena , 2002, Cell.

[50]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

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

[52]  S. Grewal,et al.  Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast. , 1998, Genetics.

[53]  R. Martienssen,et al.  Two Novel Proteins, Dos1 and Dos2, Interact with Rik1 to Regulate Heterochromatic RNA Interference and Histone Modification , 2005, Current Biology.

[54]  Michael Hampsey,et al.  Tails of Intrigue Phosphorylation of RNA Polymerase II Mediates Histone Methylation , 2003, Cell.

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

[56]  M. Keogh,et al.  Bur1 Kinase Is Required for Efficient Transcription Elongation by RNA Polymerase II , 2003, Molecular and Cellular Biology.

[57]  D. Moazed,et al.  Heterochromatin and Epigenetic Control of Gene Expression , 2003, Science.

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

[59]  C. Bonilla,et al.  RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing. , 2005, Genes & development.

[60]  T. Fujisawa,et al.  Analysis of a piwi-Related Gene Implicates Small RNAs in Genome Rearrangement in Tetrahymena , 2002, Cell.

[61]  T. Maniatis,et al.  An extensive network of coupling among gene expression machines , 2002, Nature.

[62]  A. Caudy,et al.  Functional Divergence between Histone Deacetylases in Fission Yeast by Distinct Cellular Localization and In Vivo Specificity , 2002, Molecular and Cellular Biology.

[63]  E. Nimmo,et al.  Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. , 1995, Genes & development.