Novel splicing variant of mouse Orc1 is deficient in nuclear translocation and resistant for proteasome-mediated degradation
Novel splicing variant of mouse Orc1 is deficient in nuclear translocation and resistant for proteasome-mediated degradation
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DOI：
10.1074/jbc.m413280200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Hanaoka, F
中科院分区：
文献类型：
--
作者：
Miyake, Y;Mizuno, T;Hanaoka, F
DNA复制是由复制前复合体和复制装置的逐步组装控制的。将起源识别复合体（ORC）装载到染色质上是复制前复合体组装的先决条件。为了确定哺乳动物ORC的生理功能，我们从小鼠NIH3T3细胞中克隆了ORC亚基cdna，并发现了Orc1、Orc2和Orc3的新变体形式，每种变体都来自于选择性RNA剪接。Orc1的变体Orc1B在第5外显子上缺少35个氨基酸残基；Orc2的变体Orc2B在外显子2中缺少48个氨基酸残基。在Orc3变体Orc3B中，在第15号外显子中只有1个氨基酸残基被删除。逆转录- pcr分析显示Orc1-3全长亚基Orc1A、Orc2A、Orc3A以及Orc2B、Orc3B在多种小鼠细胞系和小鼠组织中广泛表达。相比之下，Orc1B仅在胸腺和胚胎早期表达。这些Orc亚基在培养细胞中的过表达表明，Orc1A、Orc2A、Orc3A、Orc2B和Orc3B定位于细胞核，而Orc1B则只存在于细胞质中。此外，mOrc1A的35个氨基酸剪接片段与β -半乳糖苷酶的融合导致其易位到细胞核中。当Orc1B短暂表达时，其降解以蛋白酶体不依赖的方式发生，而Orc1A则通过泛素-蛋白酶体途径迅速降解。综上所述，我们得出结论，小鼠Orc1、Orc2和Orc3分别存在于两种选择性剪接变体中，而自然发生的Orc1B缺乏一个对核易位和蛋白酶体依赖性降解至关重要的功能域。
DNA replication is controlled by the stepwise assembly of the pre-replicative complex and the replication apparatus. Loading of the origin recognition complex (ORC) onto the chromatin is a prerequisite for the assembly of the pre-replicative complex. To define the physiological functions of the mammalian ORC, we cloned ORC subunit cDNAs from mouse NIH3T3 cells and found novel variant forms of Orc1, Orc2, and Orc3 each derived from alternative RNA splicing. The variant form of Orc1, Orc1B, lacks 35 amino acid residues in exon 5; the variant of Orc2, Orc2B, lacks 48 amino acid residues in exon 2. In the Orc3 variant, Orc3B, only 1 amino acid residue is deleted in exon 15. Reverse transcription-PCR analysis showed that the full-length Orc1-3 subunits, Orc1A, Orc2A, and Orc3A, as well as Orc2B and Orc3B, were widely expressed in various mouse cell lines and mouse tissues. In contrast, Orc1B was only expressed in the thymus and at an early embryonic stage. Overexpression of these Orc subunits in cultured cells revealed that Orc1A, Orc2A, Orc3A, Orc2B, and Orc3B are localized in the nucleus, whereas Orc1B remains exclusively in the cytoplasm. Moreover, fusion of the 35 amino acids spliced fragment from mOrc1A with beta-galactosidase resulted in its translocation into the nucleus. When Orc1B is expressed transiently, its degradation occurs in a proteasome-independent manner, whereas Orc1A is rapidly degraded by the ubiquitin-proteasome pathway. Taken together, we conclude that mouse Orc1, Orc2, and Orc3 each exist in two alternative-splicing variants and that naturally occurring Orc1B lacks a functional domain that is essential for nuclear translocation and proteasome-dependent degradation.