BACKGROUND Long Interspersed Element 1 (LINE-1) is a retrotransposon that is present in 500,000 copies in the human genome. Along with Alu and SVA elements, these three retrotransposons account for more than a third of the human genome sequence. These mobile elements are able to copy themselves within the genome via an RNA intermediate, a process that can promote genome instability. LINE-1 encodes two proteins, ORF1p and ORF2p. Association of ORF1p, ORF2p and a full-length L1 mRNA in a ribonucleoprotein (RNP) particle, L1 RNP, is required for L1 retrotransposition. Previous studies have suggested that fusion of a tag to L1 proteins can interfere with L1 retrotransposition. RESULTS Using antibodies detecting untagged human ORF1p, western blot analysis and manipulation of ORF1 sequence and length, we have identified a set of charged amino acids in the C-terminal region of ORF1p that are important in determining its subcellular localization. Mutation of 7 non-identical lysine residues is sufficient to make the resulting ORF1p to be predominantly cytoplasmic, demonstrating intrinsic redundancy of this requirement. These residues are also necessary for ORF1p to retain its association with KPNA2 nuclear pore protein. We demonstrate that this interaction is significantly reduced by RNase treatment. Using co-IP, we have also determined that human ORF1p associates with all members of the KPNA subfamily. CONCLUSIONS The prediction of NLS sequences suggested that specific sequences within ORF1p could be responsible for its subcellular localization by interacting with nuclear binding proteins. We have found that multiple charged amino acids in the C-terminus of ORF1p are involved in ORF1 subcellular localization and interaction with KPNA2 nuclear pore protein. Our data demonstrate that different amino acids can be mutated to have the same phenotypic effect on ORF1p subcellular localization, demonstrating that the net number of charged residues or protein structure, rather than their specific location, is important for the ORF1p nuclear localization. We also identified that human ORF1p interacts with all members of the KPNA family of proteins and that multiple KPNA family genes are expressed in human cell lines.

中文翻译：

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鉴定人 L1 ORF1 蛋白核定位所需的带电氨基酸。

背景 长散在元素 1 (LINE-1) 是一种反转录转座子，在人类基因组中存在 500,000 个拷贝。连同 Alu 和 SVA 元素，这三个反转录转座子占人类基因组序列的三分之一以上。这些移动元件能够通过 RNA 中间体在基因组内自我复制，这一过程可以促进基因组的不稳定性。LINE-1 编码两种蛋白质，ORF1p 和 ORF2p。L1 逆转录转座需要 ORF1p、ORF2p 和核糖核蛋白 (RNP) 颗粒 L1 RNP 中的全长 L1 mRNA 的关联。以前的研究表明，标签与 L1 蛋白的融合会干扰 L1 逆转录转座。结果 使用抗体检测未标记的人 ORF1p、蛋白质印迹分析和 ORF1 序列和长度的操作，我们已经在 ORF1p 的 C 末端区域鉴定出一组带电氨基酸，这些氨基酸对于确定其亚细胞定位很重要。7 个不相同的赖氨酸残基的突变足以使产生的 ORF1p 主要是细胞质的，证明了这一要求的内在冗余。这些残基也是 ORF1p 保持其与 KPNA2 核孔蛋白结合所必需的。我们证明 RNase 处理显着减少了这种相互作用。使用 co-IP，我们还确定人类 ORF1p 与 KPNA 亚家族的所有成员相关。结论 NLS 序列的预测表明，ORF1p 内的特定序列可能通过与核结合蛋白相互作用来负责其亚细胞定位。我们发现ORF1p C 末端的多个带电氨基酸参与ORF1 亚细胞定位和与KPNA2 核孔蛋白的相互作用。我们的数据表明，不同的氨基酸可以突变以对 ORF1p 亚细胞定位具有相同的表型影响，这表明带电残基或蛋白质结构的净数量，而不是它们的特定位置，对 ORF1p 核定位很重要。我们还发现人类 ORF1p 与 KPNA 蛋白质家族的所有成员相互作用，并且多个 KPNA 家族基因在人类细胞系中表达。我们的数据表明，不同的氨基酸可以突变以对 ORF1p 亚细胞定位具有相同的表型影响，这表明带电残基或蛋白质结构的净数量，而不是它们的特定位置，对 ORF1p 核定位很重要。我们还发现人类 ORF1p 与 KPNA 蛋白质家族的所有成员相互作用，并且多个 KPNA 家族基因在人类细胞系中表达。我们的数据表明，不同的氨基酸可以突变以对 ORF1p 亚细胞定位具有相同的表型影响，这表明带电残基或蛋白质结构的净数量，而不是它们的特定位置，对 ORF1p 核定位很重要。我们还发现人类 ORF1p 与 KPNA 蛋白质家族的所有成员相互作用，并且多个 KPNA 家族基因在人类细胞系中表达。