Life Sciences Center, Institute of Biotechnology, Vilnius University, Sauletekio Av. 7, 10257, Vilnius, Lithuania.
Sci Rep. 2023 Apr 14;13(1):6123. doi: 10.1038/s41598-023-32600-w.
Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like 'long' and truncated 'short' pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5'-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.
Argonaute (Ago) 蛋白存在于生命的三个领域。研究最充分的一组是真核 Ago（eAgos）。作为 RNA 干扰机制的结构核心，它们使用向导 RNA 分子进行 RNA 靶向。原核 Ago（pAgos）在结构（存在类似 eAgo 的“长”和截断的“短”pAgos）和机制方面更加多样化，因为许多 pAgos 特异性针对 DNA，而不是 RNA 向导和/或靶链。一些长 Ago 充当抗病毒防御系统。最近，短 pAgo 编码系统 SPARTA 和 GsSir2/Ago 的防御作用得到了证明，但所有其他短 pAgos 的功能和作用机制仍不清楚。在这项工作中，我们专注于由古生菌 Archaeoglobus fulgidus 编码的截断长-B Argonaute 蛋白 AfAgo 的向导和靶链偏好性。我们证明 AfAgo 与体内携带 5'-末端 AUU 核苷酸的小 RNA 分子结合，并在体外表征其与各种 RNA 和 DNA 向导/靶链的亲和力。我们还展示了与寡脱氧核苷酸 DNA 结合的 AfAgo 的 X 射线结构，为 AfAgo 与向导和靶链的碱基特异性相互作用提供了原子细节。我们的发现拓宽了目前已知的 Argonaute-核酸识别机制的范围。
