Evolutionary Conservation of the Functional Modularity of Primate and Murine LINE-1 Elements

LINE-1 (L1) retroelements emerged in mammalian genomes over 80 million years ago with a few dominant subfamilies amplifying over discrete time periods that led to distinct human and mouse L1 lineages. We evaluated the functional conservation of L1 sequences by comparing retrotransposition rates of chimeric human-rodent L1 constructs to their parental L1 counterparts. Although amino acid conservation varies from ∼35% to 63% for the L1 ORF1p and ORF2p, most human and mouse L1 sequences can be functionally exchanged. Replacing either ORF1 or ORF2 to create chimeric human-mouse L1 elements did not adversely affect retrotransposition. The mouse ORF2p retains retrotransposition-competency to support both Alu and L1 mobilization when any of the domain sequences we evaluated were substituted with human counterparts. However, the substitution of portions of the mouse cys-domain into the human ORF2p reduces both L1 retrotransposition and Alu trans-mobilization by 200–1000 fold. The observed loss of ORF2p function is independent of the endonuclease or reverse transcriptase activities of ORF2p and RNA interaction required for reverse transcription. In addition, the loss of function is physically separate from the cysteine-rich motif sequence previously shown to be required for RNP formation. Our data suggest an additional role of the less characterized carboxy-terminus of the L1 ORF2 protein by demonstrating that this domain, in addition to mediating RNP interaction(s), provides an independent and required function for the retroelement amplification process. Our experiments show a functional modularity of most of the LINE sequences. However, divergent evolution of interactions within L1 has led to non-reciprocal incompatibilities between human and mouse ORF2 cys-domain sequences.

LINE-1（L1）逆转座子于8000万年前出现于哺乳动物基因组中，少数优势亚家族在离散的时间段内发生扩增，进而形成了独特的人类与小鼠L1谱系。我们通过对比嵌合人-啮齿类L1重组构建体与其亲本L1原件的逆转座活性，评估了L1序列的功能保守性。尽管L1开放阅读框1蛋白（ORF1p）与开放阅读框2蛋白（ORF2p）的氨基酸保守性介于约35%至63%之间，但多数人类与小鼠L1序列在功能上可实现互换。替换ORF1或ORF2以构建嵌合人-小鼠L1元件，并未对逆转座活性造成不利影响。当我们所评估的任一结构域序列被替换为人类同源序列时，小鼠ORF2p仍可保留逆转座活性，以支持Alu元件（Alu）与L1的转座动员。然而，将小鼠半胱氨酸结构域（cys-domain）的部分序列替换至人类ORF2p中，会使L1逆转座活性与Alu反式转座动员能力降低200至1000倍。所观察到的ORF2p功能丧失，与ORF2p的核酸内切酶、逆转录酶活性以及逆转录过程所需的RNA相互作用均无关联。此外，该功能丧失区域与此前报道的、参与核糖核蛋白复合物（RNP）形成所需的富含半胱氨酸基序序列在物理位置上相互分离。我们的研究数据揭示了L1 ORF2蛋白中特征性较弱的羧基末端结构域的额外功能：该结构域除介导核糖核蛋白复合物相互作用外，还为逆转座因子的扩增过程提供了一项独立且必需的功能。本实验证实多数LINE序列（LINE）具备功能模块化特性，但L1内部相互作用的趋异进化，导致人类与小鼠ORF2半胱氨酸结构域序列之间存在非互作不相容性。