In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition
In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition
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DOI：
10.1073/pnas.94.19.10155
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发表时间：
1997-09-16
影响因子：
11.1
通讯作者：
Martin, SL
中科院分区：
文献类型：
--
作者：
Kolosha, VO;Martin, SL
LINE是转座元件，广泛分布于真核生物中，通过RNA中间体的逆转录进行移动。哺乳动物有两个ORFs(ORF1和ORF2)。这些ORFs编码的蛋白质在逆转录转座过程中起着重要的作用，虽然预测的ORF1的氨基酸序列与任何已知的蛋白质都没有密切的联系，但它是高度碱性的；因此，长期以来一直假设ORF1蛋白在逆转录转座过程中发挥与LINE-1(L1)RNA结合的功能。对小鼠和人胚胎癌细胞提取物中ORF1蛋白和L1RNA的共作用表明，ORF1蛋白与L1 RNA结合，形成核糖核蛋白颗粒，基于紫外光交联和使用纯化组分的电泳迁移率改变分析，我们在这里证明了由小鼠L1编码的ORF1蛋白与核酸结合，并对RNA和其他单链核酸有强烈的偏好，此外，多个拷贝的ORF1蛋白似乎以一种正的协同作用的方式与单链核酸结合；这种结合特征可能是通过免疫共沉淀检测到的ORF1多肽分子之间的蛋白质-蛋白质相互作用而促进的。这些观察结果与含有L1RNA和ORF1蛋白的核糖核蛋白颗粒的形成一致，为ORF1蛋白在L1逆转录转座中的作用提供了额外的证据。
LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process, Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle, Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids, Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein-protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.