Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center, Tulane Center for Aging, New Orleans, Louisiana, United States of America.
PLoS One. 2013 Dec 4;8(12):e82021. doi: 10.1371/journal.pone.0082021. eCollection 2013.
Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.
长散布元件 1（LINE-1，L1）是一种活跃的反转录转座子，它利用由全长双顺反子 L1 mRNA 和该 mRNA 编码的两种蛋白质（ORF1p 和 ORF2p）组成的核糖核蛋白颗粒（RNP）中间体进行移动。ORF1p 和 ORF2p 对其编码的 mRNA 表现出顺式偏好。先前对 ORF1p 的研究，从细菌和昆虫细胞中纯化，证明了这种蛋白质在体外形成三聚体。虽然这对于理解 ORF1p 功能很有价值，但这些体外方法并不能提供任何关于哺乳动物细胞中 ORF1p 自我相互作用的信息。我们使用哺乳动物双杂交（M2H）系统来研究人类和小鼠细胞中的 L1 ORF1p 自我相互作用。我们证明 M2H 系统能够成功检测瞬时转染的哺乳动物细胞中的人源和鼠源 ORF1p 自我相互作用。我们还生成了鼠源和人源 ORF1p 特异性抗体，以表征用于 M2H 系统的 ORF1p 融合蛋白的表达。使用这些抗体，我们证明 ORF1p 的反式相互作用导致异源二聚体的形成，预计会在 M2H 系统中产生阳性信号。虽然 L1 ORF1p 顺式偏好在 L1 移动中的作用已确立，但 ORF1p 反式相互作用的能力对 L1 复制周期的影响尚不清楚。此外，全长 L1、野生型 ORF1 或密码子优化的 ORF1 表达载体产生的 ORF1p 的 Western blot 分析在核中检测到。相比之下，在鼠源和人源 ORF1 蛋白的 N 端添加标签可以以标签特异性的方式显著改变亚细胞定位。这些数据支持 ORF1p 的核定位可能有助于 L1（和潜在的 SINE Alu）RNP 在宿主细胞中的核进入。
