Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs.
Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs.
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DOI：
10.1007/s00412-009-0244-2
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发表时间：
2010-04
期刊：
影响因子：
1.6
通讯作者：
Nigg EA
中科院分区：
文献类型：
--
作者：
Hübner NC;Wang LH;Kaulich M;Descombes P;Poser I;Nigg EA
DNA 依赖性腺苷三磷酸酶 (ATPase) Plk1 相互作用检查点解旋酶 (PICH) 最近与纺锤体检查点 (SAC) 信号传导有关（Baumann 等人，Cell 128(1):101–114,）。通过 siRNA 消除 PICH 会消除 SAC，并导致着丝粒中 Mad2 明显选择性丢失，表明 PICH 在 Mad1-Mad2 相互作用的调节中发挥作用。在PICH耗尽的细胞中过表达PICH对SAC功能的明显挽救最初似乎证实了PICH在SAC中的作用。然而，我们随后发现所有消除 SAC 的 PICH 导向 siRNA 寡核苷酸也会降低 Mad2 mRNA 和蛋白表达。这种减少在功能上具有重要意义，因为 PICH siRNA 不会消除含有驱动小鼠 Mad2 表达的细菌人工染色体的细胞系中的 SAC 活性。此外，我们还发现了几种 siRNA 双链体，它们可以有效地消耗 PICH，但不会显着影响 SAC 功能或 Mad2 丰度或定位。最后，我们发现过表达的 PICH 在 PICH 耗尽的细胞中恢复 SAC 活性的能力取决于有丝分裂激酶 Plk1 的隔离，而不是 PICH 的 ATP 酶活性，这表明了“旁路抑制”的潜在机制。为了支持这一观点，Plk1 的消耗或抑制也挽救了含有低水平 Mad2 的细胞中的 SAC 活性。这一观察结果表明，Plk1 活性的降低部分补偿了 Mad2 水平的降低，并认为 Plk1 通常会降低 SAC 信号传导的强度。总的来说，我们的结果质疑 PICH 在 SAC 中的作用，而是将 Mad2 确定为小 RNA 双链体的敏感脱靶。为了支持后一个结论，我们的证据表明，Mad2 的脱靶效应也可能有助于解释 Tai1 激酶在 SAC 信号传导中的明显作用（Draviam 等人，Nat Cell Biol 9(5):556–564,）。本文的在线版本 (doi:10.1007/s00412-009-0244-2) 包含补充材料，可供授权用户使用。
The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101–114,). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1–Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of “bypass suppression.” In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling (Draviam et al., Nat Cell Biol 9(5):556–564,). The online version of this article (doi:10.1007/s00412-009-0244-2) contains supplementary material, which is available to authorized users.
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