Nucleic acids copurified with dAmAgo from E coli cells. Escherichia coli str. K-12 substr. MG1655

Argonautes are an evolutionary conserved family of programmable nucleases that identify target nucleic acids using small guide oligonucleotides. In contrast to eukaryotic Argonautes (eAgos) that act on RNA, most prokaryotic Argonautes (pAgos) recognize DNA targets. Similarly to eAgos, pAgos can protect prokaryotic cells from invaders, but the biogenesis of guide oligonucleotides that confer them specificity to their targets remains poorly understood. Here, we have identified a new group of RNA-guided pAgo nucleases and demonstrated that a representative pAgo from this group, AmAgo from the mesophilic bacterium Alteromonas macleodii, binds guide RNAs of varying lengths for specific DNA targeting. Unlike most pAgos and eAgos, AmAgo is strictly specific to unphosphorylated RNA guides containing a 5-adenosine. AmAgo and related pAgos are co-encoded with a conserved protein, Agap, which we characterized as an RNA endonuclease from the HEPN superfamily. In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing unphosphorylated 5-A guide oligonucleotides bound by AmAgo. In vivo, Agap cooperates with AmAgo in acquiring guide RNAs and counteracting bacteriophage infection. The AmAgo-Agap pair represents the first example of a pAgo system that autonomously produces RNA guides for DNA targeting and antiviral defense, which holds promise for programmable DNA targeting in biotechnology.

Argonaute蛋白家族（Argonautes）是一类进化保守的可编程核酸酶，可通过小型向导寡核苷酸（small guide oligonucleotides）识别靶核酸。与作用于RNA的真核Argonaute蛋白（eukaryotic Argonautes, eAgos）不同，大多数原核Argonaute蛋白（prokaryotic Argonautes, pAgos）识别DNA靶标。与真核Argonaute蛋白类似，原核Argonaute蛋白可保护原核细胞免受外源入侵者侵害，但赋予其靶标特异性的向导寡核苷酸的生物发生过程仍尚未被充分阐明。本研究鉴定出一类新型RNA向导型原核Argonaute核酸酶，并证实该家族中的代表性成员——来自嗜温性细菌交替单胞菌（Alteromonas macleodii）的AmAgo——可结合不同长度的向导RNA以实现特异性DNA靶向。与大多数原核和真核Argonaute蛋白不同，AmAgo对携带5'腺苷的未磷酸化RNA向导具有严格的结合特异性。AmAgo及其相关原核Argonaute蛋白与一类保守蛋白Agap共编码，我们将该蛋白鉴定为HEPN超家族的RNA核酸内切酶。体外实验表明，Agap可在鸟苷酸与腺苷酸之间切割RNA，生成被AmAgo结合的未磷酸化5'端腺苷向导寡核苷酸。体内实验中，Agap与AmAgo协同发挥功能，参与向导RNA的获取并抵御噬菌体感染。AmAgo-Agap配对系统是首个可自主产生用于DNA靶向的RNA向导并发挥抗病毒防御功能的原核Argonaute系统，其在生物技术领域的可编程DNA靶向应用中具有广阔前景。