Iida Tetsushi, Nakayama Jun-ichi, Moazed Danesh
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Mol Cell. 2008 Jul 25;31(2):178-89. doi: 10.1016/j.molcel.2008.07.003.
Heterochromatic gene silencing at the pericentromeric DNA repeats in fission yeast requires the RNA interference (RNAi) machinery. The RNA-induced transcriptional silencing (RITS) complex mediates histone H3 lysine 9 (H3K9) methylation and recruits the RNA-dependent RNA polymerase complex (RDRC) to promote double-stranded RNA (dsRNA) synthesis and siRNA generation. Here we show that ectopic expression of a long hairpin RNA bypasses the requirement for chromatin-dependent steps in siRNA generation. The ability of hairpin-produced siRNAs to silence homologous sequences in trans is subject to local chromatin structure, requires HP1, and correlates with antisense transcription at the target locus. Furthermore, although hairpin siRNAs can be produced in the absence of RDRC, trans-silencing of reporter genes by hairpin-produced siRNAs is completely dependent on the dsRNA synthesis activity of RDRC. These results provide insights into the regulation of siRNA action and reveal roles for cis-dsRNA synthesis and HP1 in siRNA-mediated heterochromatin assembly.
裂殖酵母着丝粒周围DNA重复序列的异染色质基因沉默需要RNA干扰（RNAi）机制。RNA诱导的转录沉默（RITS）复合物介导组蛋白H3赖氨酸9（H3K9）甲基化，并招募RNA依赖性RNA聚合酶复合物（RDRC）以促进双链RNA（dsRNA）合成和小干扰RNA（siRNA）生成。我们在此表明，长发夹RNA的异位表达绕过了siRNA生成中对染色质依赖性步骤的需求。发夹产生的siRNA在反式作用中沉默同源序列的能力受局部染色质结构影响，需要HP1，并与靶位点的反义转录相关。此外，尽管发夹siRNA可以在没有RDRC的情况下产生，但发夹产生的siRNA对报告基因的反式沉默完全依赖于RDRC的dsRNA合成活性。这些结果为siRNA作用的调控提供了见解，并揭示了顺式dsRNA合成和HP1在siRNA介导的异染色质组装中的作用。
