Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast.
RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double-stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. Here, we show that green fluorescent protein (GFP) hairpin dsRNA allows production of high levels of Argonaute-associated small interfering RNAs (siRNAs), which can induce heterochromatin formation at a remote locus. This silencing does not require any manipulation apart from hairpin expression. In cells expressing a ura4(+)-GFP fusion gene, production of GFP siRNAs causes the appearance of ura4 siRNAs from the target gene. Production of these secondary siRNAs depends on RNA-dependent RNA polymerase Rdp1 (RDRP(Rdp1)) function and other RNAi pathway components. This demonstrates that transitivity occurs in fission yeast and implies that RDRP(Rdp1) can synthesize RNA from targeted RNA templates in vivo, generating siRNAs not homologous to the hairpin.
Simmer
Femke
F
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, 6.34 Swann Building, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.
Buscaino
Alessia
A
Kos-Braun
Isabelle C
IC
Kagansky
Alexander
A
Boukaba
Abdelhalim
A
Urano
Takeshi
T
Kerr
Alastair R W
AR
Allshire
Robin C
RC
D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006570:Heterochromatin / Q000235:genetics / Q000378:metabolism; D055029:Inverted Repeat Sequences / Q000502:physiology; D030781:Organisms, Genetically Modified; D012313:RNA / Q000737:chemistry / Q000502:physiology*; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D012568:Schizosaccharomyces / Q000235:genetics*
D006570:Heterochromatin; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D012313:RNA