Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis
Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis
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DOI：
10.1073/pnas.1816652116
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发表时间：
2019-02-26
影响因子：
11.1
通讯作者：
Zheng，Binglian
中科院分区：
文献类型：
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作者：
Zhong，Songxiao;Xu，Yifeng;Zheng，Binglian
在RNA导向的DNA甲基化(RdDM)过程中，由DRD1、DMS3和RDM1组成的DDR复合体负责招募DNA聚合酶V(PolV)来沉默植物中的转座元件(TES)。然而，DDR复合体是如何受到监管的仍未被探索。在这里，我们证明了后期促进复合体/环体(APC/C)通过靶向DMS3降解来调节DDR复合体的组装。我们发现，在apc/andpolv突变体中，大量的RdDM基因座通常是去抑制的，RdDM活性的缺陷是由于DMS3蛋白水平上调，最终导致Pol V募集减少。DMS3被APC/C泛素化，以D盒依赖的方式降解。竞争结合分析和凝胶过滤分析表明，适当水平的DMS3对DDR复合体的组装至关重要。与DMS3水平的重要性一致，DMS3的过度积累导致RdDM活性缺陷，表现为apc/anddms3突变体。此外，DMS3的表达依赖于细胞周期。总而言之，这些发现为DDR复合体的组装如何受到调控提供了直接证据，并揭示了APC/C在调控RdDM活动中的保护作用。
During RNA-directed DNA methylation (RdDM), the DDR complex, composed of DRD1, DMS3, and RDM1, is responsible for recruiting DNA polymerase V (Pol V) to silence transposable elements (TEs) in plants. However, how the DDR complex is regulated remains unexplored. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) regulates the assembly of the DDR complex by targeting DMS3 for degradation. We found that a substantial set of RdDM loci was commonly de-repressed inapc/candpol vmutants, and that the defects in RdDM activity resulted from up-regulated DMS3 protein levels, which finally caused reduced Pol V recruitment. DMS3 was ubiquitinated by APC/C for degradation in a D box-dependent manner. Competitive binding assays and gel filtration analyses showed that a proper level of DMS3 is critical for the assembly of the DDR complex. Consistent with the importance of the level of DMS3, overaccumulation of DMS3 caused defective RdDM activity, phenocopying theapc/canddms3mutants. Moreover, DMS3 is expressed in a cell cycle-dependent manner. Collectively, these findings provide direct evidence as to how the assembly of the DDR complex is regulated and uncover a safeguarding role of APC/C in the regulation of RdDM activity.