Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization
Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization
复制标题
DOI：
10.1128/mcb.19.12.8536
复制
发表时间：
1999-12
影响因子：
5.3
通讯作者：
Hung-Kai Chen;C. Pai;Jingrong Huang;N. Yeh
中科院分区：
文献类型：
--
作者：
Hung-Kai Chen;C. Pai;Jingrong Huang;N. Yeh
摘要 Nopp140 被认为在核仁和细胞质之间穿梭。然而，来自不同物种的 Nopp140 同源物的主要核仁定位表明 Nopp140 也参与核仁内发生的事件。在这项研究中，我们证明了 RNA 聚合酶 I 的最大亚基 RPA194 与人 Nopp140 (hNopp140) 进行了免疫共沉淀。这种相互作用是通过 hNopp140 的氨基酸 204 至 382 介导的。通过双重免疫荧光，hNopp140 与 RNA 聚合酶 I 共定位于核仁中的 rDNA（rRNA 基因）转录活性位点。这些结果表明 Nopp140 可以在体内与 RNA 聚合酶 I 相互作用。表达 hNopp140 氨基末端一半 hNopp140N382（氨基酸 1 至 382）的转染细胞显示出具有新月形结构的改变的核仁。这种表型让人想起放线菌素 D 处理诱导的分离核仁，已知放线菌素 D 可以抑制 rRNA 合成。原位连续检测显示，hNopp140N382 蛋白错误定位了内源性 RNA 聚合酶 I，并关闭了细胞 rRNA 基因转录。突变体 hNopp140N382 的这些显性负效应表明 Nopp140 在 rDNA 转录中发挥重要作用。有趣的是，hNopp140 的异位表达达到非常高的水平，导致形成转录失活的球形结构，占据整个核仁区域，捕获 RNA 聚合酶 I、fibrillarin 和 hNopp140，但排除核仁素。 hNopp140 过表达后这些核仁蛋白的错误定位意味着 Nopp140 也可能在维持核仁完整性中发挥作用。
ABSTRACT Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.