Thawani Akanksha, Rodríguez-Vargas Anthony, Van Treeck Briana, Hassan Nozhat T, Adelson David L, Nogales Eva, Collins Kathleen
California Institute for Quantitative Biosciences (QB3), Berkeley, CA, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Sci Adv. 2025 Jun 20;11(25):eadu5533. doi: 10.1126/sciadv.adu5533.
R2 retrotransposons are site-specific eukaryotic non-long terminal repeat retrotransposons that copy and paste into gene loci encoding ribosomal RNAs. Recently, we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not well characterized. Here, we report high-resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins at the initiation of target-primed reverse transcription and after cDNA synthesis and second-strand nicking. Using biochemical and cellular assays, we illuminate the basis for high selectivity of template use and unique roles for each of the three zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif specific to vertebrate A-clade R2 proteins. Our work provides the first insights into A-clade R2 protein structure during gene insertion and may enable future improvement and adaptation of R2-based systems for precise transgene insertion.
R2反转录转座子是位点特异性的真核生物非长末端重复反转录转座子，可复制并粘贴到编码核糖体RNA的基因座中。最近，我们证明了鸟类A类R2蛋白能够将转基因高效且精确地插入人类细胞中的天然安全港位点。支持基因插入的A类R2蛋白的特征尚未得到充分表征。在此，我们报告了两种脊椎动物A类R2蛋白在靶标引发的逆转录起始时以及cDNA合成和第二链切口后的高分辨率冷冻电子显微镜结构。通过生化和细胞分析，我们阐明了模板使用高选择性的基础以及三个锌指结构域在核酸识别中的独特作用。脊椎动物A类R2蛋白特有的意外插入基序增强了逆转录酶活性位点结构。我们的工作首次揭示了基因插入过程中A类R2蛋白的结构，并可能使基于R2的系统在未来得到改进和适应，以实现精确的转基因插入。
