CREST Research Program, Japan Science and Technology Corporation, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Japan.
PLoS One. 2007 Mar 21;2(3):e317. doi: 10.1371/journal.pone.0000317.
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.
细胞 RNA 代谢在细胞生长和分裂中有广泛的功能方面，但在有丝分裂过程中染色体分离的作用知之甚少。Dis3 核糖核酸酶是 RNA 加工外切体复合物的关键组成部分。先前分离的裂殖酵母 Schizosaccharomyces pombe 的 dis3-54 冷敏感突变体表明，Dis3 也需要正确的染色体分离。
方法/主要发现：我们在这里表明，dis3-54 中的有丝分裂进程被阻断，并且染色体的分离被有丝分裂检查点控制的激活所阻断。这种阻断依赖于 Mad2 检查点蛋白。双突变体和抑制剂分析表明，Dis3 是正确的动粒形成和功能所必需的，并且 Mad2 检查点监测这种活性。Dis3 是高度保守的 RNase II 家族的成员，并且已知是外切体复合物的必需亚基。发现 dis3-54 突变改变了 Dis3 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重复（otr）和中央核心（cnt 和 imr）区域的 ura4(+)报告基因的沉默丢失有关。另一方面，在 dis3-54 突变体中，着丝粒的 siRNA 成熟和 RITS RNAi 效应物复合物的形成是正常的。微球菌核酸酶测定也表明，dis3-54 突变体中的着丝粒的整体染色质结构没有受到影响。
结论/意义：发现外切体核心亚基 Dis3 的核糖核酸酶活性对于正确的动粒形成和建立动粒-微管相互作用是必需的。此外，Dis3 被认为通过参与外着丝粒重复和中央核心区域内的异染色质沉默对动粒形成作出贡献。这种活性可能受到有丝分裂检查点的监测，并且与直接靶向外着丝粒重复的 RNAi 介导的异染色质形成不同。
