Background In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ 861393-28-4 supplier domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically 861393-28-4 supplier associated with genes for 861393-28-4 supplier other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system 861393-28-4 supplier targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain. Conclusion The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests. Reviewers This article was reviewed by Daniel Haft, 861393-28-4 supplier Martijn Huynen, and Chris Ponting. Background The discovery of elaborate and versatile systems of RNA-mediated gene silencing in eukaryotes is one of the pivotal advances in biology of RICTOR the last decade [1-5]. There are three major, distinct forms of regulatory small RNAs involved in eukaryotic gene silencing: small interfering (si) RNAs, micro (mi) RNAs, and PIWI-associated (pi) RNA (previously referred to as rasiRNA) [6]. The siRNAs are derived from double-stranded RNAs of viruses and transposable elements, which are processed by Dicer, one of the essential components of the RNA-Induced Silencing Complexes (RISCs) [7-11]. Dicer cleaves long dsRNA molecules into short, 21C22 nucleotide duplexes which are subsequently unwound and the guide strand is loaded on another crucial component of RISC, the Argonaute (Ago) slicer nuclease. The Ago-siRNA complex then binds to the target mRNA which is cleaved by the PIWI domain of Argonaute (Ago), after which the mRNA fragments are released and the RISC-siRNA catalytic complex is recycled [9,12-14]. Variant, paralogous Dicers and Argonautes are involved in the mechanisms of.