Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon
Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon
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DOI：
10.1093/emboj/16.19.6034
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发表时间：
1997-10-01
期刊：
影响因子：
11.4
通讯作者：
Singer, MF
中科院分区：
文献类型：
--
作者：
Hohjoh, H;Singer, MF
先前使用人畸胎癌细胞的实验表明，p40（由人 LINE-1 (L1Hs) 逆转录转座子的第一个开放阅读框 (ORF) 编码的蛋白质）存在于与 L1Hs RNA（p40 RNP 复合物）直接相关的大型细胞质核糖核蛋白复合物中。我们现在已经使用依赖于 p40 和结合 RNA 的免疫共沉淀的 RNA 结合测定在体外研究了部分纯化的 p40 和 L1Hs RNA 之间的相互作用。这些实验鉴定了全长有义链 L1Hs RNA 上的两个 p40 结合位点。这两个位点均位于 6000 nt RNA 的第二个 ORF 中：位点 A 位于残基 1999 和 2039 之间，位点 B 位于残基 4839 和 4875 之间。这两个 RNA 片段共享同源区域。涉及 UV 交联和免疫沉淀的实验表明，体外复合物中的 p40 与 L1Hs RNA 直接相关，就像在畸胎癌细胞中发现的 p40 RNP 复合物中一样。结合和竞争实验表明，p40 与含有 p40 结合位点的单链 RNA 结合，但不与单链或双链 DNA、双链 RNA 或含有结合位点序列的 DNA-RNA 杂合体结合。因此，p40 似乎是一种序列特异性的单链 RNA 结合蛋白。
Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE-1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co-immunoprecipitation of p40 and bound RNA, These experiments identified two p40 binding sites on the full-length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross-linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single-stranded RNA containing a p40 binding site, but not to single-stranded or double-stranded DNA, double-stranded RNA or a DNA-RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence-specific, single-strand RNA binding protein.