Wellcome Trust Centre for Cell Biology, School of Biological Sciences, 6.34 Swann Building, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.
EMBO Rep. 2010 Feb;11(2):112-8. doi: 10.1038/embor.2009.273. Epub 2010 Jan 8.
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.
RNA 干扰 (RNAi) 在真核生物中广泛存在，并通过转录或转录后调控基因表达。在裂殖酵母中，RNAi 与模板转录紧密偶联，并与建立顺式异染色质的染色质修饰有关。外源性双链 RNA (dsRNA) 似乎只有在某些沉默蛋白过表达时才能诱导转座体异染色质的形成。在这里，我们表明绿色荧光蛋白 (GFP) 发夹 dsRNA 允许产生高水平的 Argonaute 相关小干扰 RNA (siRNA)，这些 siRNA 可以在远程基因座诱导异染色质的形成。这种沉默除了发夹表达外不需要任何操作。在表达 ura4(+)-GFP 融合基因的细胞中，GFP siRNA 的产生导致靶基因的 ura4 siRNA 的出现。这些二级 siRNA 的产生依赖于 RNA 依赖性 RNA 聚合酶 Rdp1（RDRP(Rdp1)）功能和其他 RNAi 途径成分。这表明转导性发生在裂殖酵母中，并暗示 RDRP(Rdp1) 可以在体内从靶向 RNA 模板合成 RNA，产生与发夹不同源的 siRNA。
