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.
bioRxiv. 2024 Nov 20:2024.11.11.623112. doi: 10.1101/2024.11.11.623112.
R2 retrotransposons are model site-specific eukaryotic non-LTR 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 characterized. Here, we report high resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins, avian and testudine, at the initiation of target-primed reverse transcription and one structure after cDNA synthesis and second strand nicking. Using biochemical and cellular assays we discover the basis for high selectivity of template use and unique roles for each of the expanded A-clade zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif in vertebrate A-clade R2 proteins. Our work brings first insights to A-clade R2 protein structure during gene insertion and enables further improvement and adaptation of R2-based systems for precise transgene insertion.
R2反转录转座子是位点特异性真核非LTR反转录转座子的模型，它们通过复制粘贴的方式插入编码核糖体RNA的基因位点。最近我们证明，鸟类A类R2蛋白能够在人类细胞中将转基因高效且精确地插入其天然的安全港位点。支持基因插入的A类R2蛋白的特征尚未得到表征。在此，我们报告了两种脊椎动物A类R2蛋白（鸟类和龟类）在靶标引发的逆转录起始时的高分辨率冷冻电子显微镜结构，以及一种在cDNA合成和第二链切口后的结构。通过生化和细胞分析，我们发现了模板使用高选择性的基础，以及每个扩展的A类锌指结构域在核酸识别中的独特作用。脊椎动物A类R2蛋白中一个意外的插入基序增强了逆转录酶活性位点的结构。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，并有助于进一步改进和适配基于R2的系统以实现精确的转基因插入。
