Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25
Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25
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DOI：
10.1016/j.yexcr.2006.02.002
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发表时间：
2006-06-10
影响因子：
3.7
通讯作者：
Tanaka, Junya
中科院分区：
文献类型：
--
作者：
Fujiwara, Takashi;Suzuki, Shunji;Tanaka, Junya
Nop25是一种被认为与rRNA转录相关的RNA结合核仁蛋白。本研究旨在确定Nop25在核仁中的定位机制。Nop25氨基酸序列的缺失实验表明，Nop25在n端含有一个核靶向序列，在c端含有一个核仁靶向序列。通过在细胞中表达来自c末端的衍生肽作为gfp融合蛋白，赖氨酸和精氨酸残基富集肽（KRKHPRRAQDSTKKPPSATRTSKTQRRRR）允许gfp融合蛋白被运输并完全保留在核仁中。当肽与cMyc表位融合并在细胞中表达时，在核仁中检测到cMyc表位。通过删除Nop25的肽，Nop25不定位于核仁。此外，在该亚结构域的赖氨酸和精氨酸残基的肽或氨基酸替换中，缺失一个亚结构域（KRKHPRRAQ）导致Nop25核仁定位的丢失。这些结果表明，赖氨酸和精氨酸残基富集肽是Nop25最突出的核仁靶向序列，碱性残基的长链可能在Nop25的核仁定位中起重要作用。虽然Nop25含有假定的SUMOylation，磷酸化和糖基化位点，但这些位点的氨基酸取代对核仁定位没有影响，因此这些翻译后修饰与Nop25在核仁中的定位无关。这些细胞表达了一种gfp融合蛋白，其核仁靶向序列为Nop25，用rna酶a处理后，导致该蛋白从核仁上完全脱位。这些数据表明，核仁靶向序列可能在Nop25与RNA分子的结合中起重要作用，并且Nop25的RNA结合可能是Nop25核仁定位的必要条件。(c) 2006爱思唯尔公司版权所有。
Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, a cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25. (c) 2006 Elsevier Inc. All rights reserved.