Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Nucleic Acids Res. 2024 Jun 24;52(11):6571-6585. doi: 10.1093/nar/gkae194.
Eukaryotic retrotransposons encode a reverse transcriptase that binds RNA to template DNA synthesis. The ancestral non-long terminal repeat (non-LTR) retrotransposons encode a protein that performs target-primed reverse transcription (TPRT), in which the nicked genomic target site initiates complementary DNA (cDNA) synthesis directly into the genome. The best understood model system for biochemical studies of TPRT is the R2 protein from the silk moth Bombyx mori. The R2 protein selectively binds the 3' untranslated region of its encoding RNA as template for DNA insertion to its target site in 28S ribosomal DNA. Here, binding and TPRT assays define RNA contributions to RNA-protein interaction, template use for TPRT and the fidelity of template positioning for TPRT cDNA synthesis. We quantify both sequence and structure contributions to protein-RNA interaction. RNA determinants of binding affinity overlap but are not equivalent to RNA features required for TPRT and its fidelity of template positioning for full-length TPRT cDNA synthesis. Additionally, we show that a previously implicated RNA-binding protein surface of R2 protein makes RNA binding affinity dependent on the presence of two stem-loops. Our findings inform evolutionary relationships across R2 retrotransposon RNAs and are a step toward understanding the mechanism and template specificity of non-LTR retrotransposon mobility.
真核逆转录转座子编码一种逆转录酶，该酶能将 RNA 结合到模板 DNA 合成上。祖先的非长末端重复（non-LTR）逆转录转座子编码一种蛋白质，该蛋白质能执行靶向引物逆转录（TPRT），其中缺口的基因组靶位点直接将互补 DNA（cDNA）合成到基因组中。在生化研究中，TPRT 的最佳研究模型系统是丝蛾 Bombyx mori 的 R2 蛋白。R2 蛋白选择性地结合其编码 RNA 的 3' 非翻译区作为 DNA 插入其靶位点 28S 核糖体 DNA 的模板。在这里，结合和 TPRT 测定定义了 RNA 对 RNA-蛋白相互作用、模板用于 TPRT 和 TPRT cDNA 合成中模板定位保真度的贡献。我们定量了序列和结构对蛋白-RNA 相互作用的贡献。结合亲和力的 RNA 决定因素重叠，但不等同于 TPRT 和其全长 TPRT cDNA 合成中模板定位保真度所需的 RNA 特征。此外，我们还表明，先前涉及的 R2 蛋白的 RNA 结合蛋白表面使 RNA 结合亲和力依赖于两个茎环的存在。我们的研究结果提供了跨越 R2 逆转录转座子 RNA 的进化关系信息，并朝着理解非 LTR 逆转录转座子运动的机制和模板特异性迈出了一步。
