RESUMO
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 studied 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 hydroxylated RNA guides containing a 5'-adenosine. AmAgo and related pAgos are co-encoded with a conserved RNA endonuclease from the HEPN superfamily (Ago-associated protein, Agap-HEPN). In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing hydroxylated 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.
Assuntos
Alteromonas , Proteínas Argonautas , DNA Viral , RNA Guia de Sistemas CRISPR-Cas , Ribonucleases , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ribonucleases/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Alteromonas/enzimologia , Alteromonas/virologia , DNA Viral/metabolismo , Bacteriófagos/fisiologiaRESUMO
Argonaute nucleases use small nucleic acid guides to recognize and degrade complementary nucleic acid targets. Most prokaryotic Argonautes (pAgos) recognize DNA targets and may play a role in cell immunity against invader genetic elements. We have recently described two related groups of pAgo nucleases that have distinct specificity for DNA guides and RNA targets (DNA>RNA pAgos). Here, we describe additional pAgos from the same clades of the pAgo tree and demonstrate that they have the same unusual nucleic acid specificity. The two groups of DNA>RNA pAgos have non-standard guide-binding pockets in the MID domain and differ in the register of guide DNA binding and target cleavage. In contrast to other pAgos, which coordinate the 5'-end of the guide molecule by their C-terminal carboxyl, DNA>RNA pAgos have an extended C-terminus located away from the MID pocket. We show that modifications of the C-terminus do not affect guide DNA binding, but inhibit cleavage of complementary and mismatched RNA targets by some DNA>RNA pAgos. Our data suggest that the unique C-terminus found in DNA>RNA pAgos can modulate their catalytic properties and can be used as a target for pAgo modifications.