R2非长末端重复反转录转座子在广泛的多细胞真核生物中位点特异性插入核糖体RNA基因(rDNA)。可以利用R2编码的蛋白质来介导培养的人细胞中28SrDNA基因座处的转基因插入。这一战略,精确的RNA介导的转基因序列(PRINT),依赖于编码R2蛋白的mRNA和编码选择的转基因盒的RNA模板的共递送。在这里,我们演示了PRINTRNA模板5'模块,作为互补的DNA3'末端将产生与rDNA的转基因5'连接,影响基因插入的效率和机制。迭代设计和测试确定了最佳的5个模块,由具有高热力学稳定性的丁型肝炎病毒样核酶折叠组成,这表明RNA模板从其5端降解可能会限制转基因插入效率。我们还证明了转基因5'连接的形成可以是精确的,通过将第一链互补DNA的3'末端与上游靶位点退火形成,或者不精确,通过末端连接,但是结形成机制的这种差异并不是插入效率的主要决定因素。不精确的末端连接事件的序列表征令人惊讶地表明对微同源性的最小依赖。我们的发现扩展了目前对R2逆转录转座子转录序列和结构的作用的理解。尤其是核酶5折叠,用于细胞中的反转录转座子迁移率和RNA模板基因合成。
R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate
transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a
transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5\' module, which as a complementary DNA 3\' end will generate the
transgene 5\' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5\' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5\' end may limit
transgene insertion efficiency. We also demonstrate that
transgene 5\' junction formation can be either precise, formed by annealing the 3\' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5\' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.