Department of Cell Biology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.
Mol Cell. 2010 Aug 13;39(3):360-72. doi: 10.1016/j.molcel.2010.07.017.
In fission yeast, the pericentromeric dg and dh repeats are transcribed and give rise to small interfering RNAs (siRNAs) by a mechanism that depends on the Clr4(suv39h) histone H3 lysine 9 (H3K9) methyltransferase. Here, we show that Clr4 activity promotes the assembly of a tripartite complex composed of the Clr4-containing CLRC complex and complexes involved in siRNA generation. However, unlike dh siRNAs, dg siRNAs accumulate to near wild-type levels in cells with H3K9 substitutions that cannot be methylated. Thus, Clr4 activity controls siRNA amplification from the different repeat regions by different mechanisms, H3K9 methylation dependent versus independent. Furthermore, artificial tethering of Rik1, a core subunit of the CLRC complex, to a euchromatic RNA mediates RNAi-dependent silencing that partially bypasses the requirement for other CLRC subunits. These findings establish Rik1 as a key link between CLRC and RNAi and reveal distinct centromeric siRNA amplification mechanisms that depend on the Clr4 methyltransferase activity.
在裂殖酵母中，着丝粒周围的 dg 和 dh 重复序列被转录，并通过一种依赖于 Clr4(suv39h)组蛋白 H3 赖氨酸 9（H3K9）甲基转移酶的机制产生小干扰 RNA（siRNA）。在这里，我们表明 Clr4 活性促进了由包含 Clr4 的 CLRC 复合物和参与 siRNA 生成的复合物组成的三分体复合物的组装。然而，与 dh siRNA 不同，dg siRNA 在 H3K9 取代不能被甲基化的细胞中积累到接近野生型水平。因此，Clr4 活性通过不同的机制控制来自不同重复区域的 siRNA 扩增，即 H3K9 甲基化依赖性和非依赖性。此外，人工将 CLRC 复合物的核心亚基 Rik1 连接到常染色质 RNA 上，可以介导 RNAi 依赖性沉默，部分绕过对其他 CLRC 亚基的需求。这些发现确立了 Rik1 作为 CLRC 和 RNAi 之间的关键联系，并揭示了依赖于 Clr4 甲基转移酶活性的不同着丝粒 siRNA 扩增机制。
