Ribonuclease Activity of Dis3 Is Required for Mitotic Progression and Provides a Possible Link between Heterochromatin and Kinetochore Function
Ribonuclease Activity of Dis3 Is Required for Mitotic Progression and Provides a Possible Link between Heterochromatin and Kinetochore Function
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DOI：
10.1371/journal.pone.0000317
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发表时间：
2007-03-21
期刊：
影响因子：
3.7
通讯作者：
Yanagida, Mitsuhiro
中科院分区：
文献类型：
--
作者：
Murakami, Hiroaki;Goto, Derek B.;Yanagida, Mitsuhiro
背景细胞RNA代谢在细胞生长和分裂中具有广泛的功能方面，但其在有丝分裂期间染色体分离中的作用知之甚少。Dis 3核糖核酸酶是RNA加工外泌体复合物的关键组分。先前分离的裂殖酵母裂殖酵母的dis 3 -54冷敏感突变体表明，Dis 3也是正确的染色体分离所必需的。方法/主要调查结果。我们在这里表明，有丝分裂的进展是在dis 3 -54逮捕，并有丝分裂检查点控制激活的染色体的分离被阻止。这种阻断依赖于Mad 2检查点蛋白。双突变体和抑制剂分析表明，Dis 3是正确的动粒形成和功能所必需的，并且这种活性由Mad 2检查点监测。Dis 3是高度保守的RNase II家族的成员，并且已知是外泌体复合物的必需亚基。发现dis 3 -54突变改变了Dis 3的RNaseII结构域，这导致体外核糖核酸酶活性降低。这与插入到着丝粒的外部重复序列（otr）和中心核心（cnt和imr）区域的ura 4（+）报告基因的沉默丢失有关。另一方面，在dis 3 -54突变体中，着丝粒siRNA成熟和RITS RNAi效应复合物的形成是正常的。微球菌核酸酶分析也表明，在dis 3 -54突变体中，着丝粒的整体染色质结构没有受到影响。结论/意义。外泌体的核心亚基Dis 3的RNA酶活性被发现是适当的动粒形成和建立动粒-微管相互作用所必需的。此外，Dis 3被认为有助于动粒的形成，通过参与异染色质沉默在两个外部着丝粒重复和中央核心区域内。这种活性可能由有丝分裂检查点监测，并且与直接靶向外部着丝粒重复的RNAi介导的异染色质形成不同。
Background. Cellular RNA metabolism has a broad range of functional aspects in cell growth and division, but its role in chromosome segregation during mitosis is only poorly understood. The Dis3 ribonuclease is a key component of the RNA-processing exosome complex. Previous isolation of the dis3-54 cold-sensitive mutant of fission yeast Schizosaccharomyces pombe suggested that Dis3 is also required for correct chromosome segregation. Methodology/Principal Findings. We show here that the progression of mitosis is arrested in dis3-54, and that segregation of the chromosomes is blocked by activation of the mitotic checkpoint control. This block is dependent on the Mad2 checkpoint protein. Double mutant and inhibitor analyses revealed that Dis3 is required for correct kinetochore formation and function, and that this activity is monitored by the Mad2 checkpoint. Dis3 is a member of the highly conserved RNase II family and is known to be an essential subunit of the exosome complex. The dis3-54 mutation was found to alter the RNaseII domain of Dis3, which caused a reduction in ribonuclease activity in vitro. This was associated with loss of silencing of an ura4(+) reporter gene inserted into the outer repeats (otr) and central core (cnt and imr) regions of the centromere. On the other hand, centromeric siRNA maturation and formation of the RITS RNAi effector complex was normal in the dis3-54 mutant. Micrococcal nuclease assay also suggested the overall chromatin structure of the centromere was not affected in dis3-54 mutant. Conclusions/Significance. RNase activity of Dis3, a core subunit of exosome, was found to be required for proper kinetochore formation and establishment of kinetochore-microtubule interactions. Moreover, Dis3 was suggested to contribute to kinetochore formation through an involvement in heterochromatic silencing at both outer centromeric repeats and within the central core region. This activity is likely monitored by the mitotic checkpoint, and distinct from that of RNAi-mediated heterochromatin formation directly targeting outer centromeric repeats.