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.
Bühler
Marc
M
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Verdel
André
A
Moazed
Danesh
D
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D006570:Heterochromatin / Q000235:genetics*; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D012322:RNA Precursors / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D037761:Sirtuins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D006570:Heterochromatin; D006657:Histones; D012322:RNA Precursors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D029702:Schizosaccharomyces pombe Proteins; C465958:Swi6 protein, S pombe; C499720:Tas3 protein, S pombe; C400810:ura4 protein, S pombe; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; C112744:clr4 protein, S pombe; C511955:Eri1 protein, S pombe; D005095:Exoribonucleases; D037761:Sirtuins