DNA interference and beyond: structure and functions of prokaryotic Argonaute proteins

Recognition and repression of RNA targets by Argonaute proteins guided by small RNAs is the essence of RNA interference in eukaryotes. Argonaute proteins with diverse structures are also found in many bacterial and archaeal genomes. Recent studies revealed that, similarly to their eukaryotic counterparts, prokaryotic Argonautes (pAgos) may function in cell defense against foreign genetic elements but, in contrast, preferably act on DNA targets. Many crucial details of the pAgo action, and the roles of a plethora of pAgos with non-conventional architecture remain unknown. Here, we review available structural and biochemical data on pAgos and discuss their possible functions in host defense and other genetic processes in prokaryotic cells

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Members of the conserved Argonaute (Ago) protein family provide defense against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases, however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with nonspecific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus, however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance

Insights into genomic DNA sampling by prokaryotic Argonaute proteins

Prokaryotic Argonaute proteins (pAgos) are endonucleases that bind small DNA or RNA guides and mediate cleavage of complementary targets. They are encoded in a variety of bacterial and archaeal genomes and supposedly participate in cell defence against foreign DNA. Previous biochemical and structural studies have elucidated the mechanistic aspects of guide binding, target search and cleavage by pAgos. pAgos have been shown to interfere with plasmid uptake in vivo and to autonomously produce guides from double-stranded DNA substrates in vitro. However, the principles underlying self/nonself discrimination remain unknown. Here we characterize in vivo guide biogenesis by pAgos from mesophilic bacteria Limnothrix rosea (LrAgo) and Clostridium butyricum (CbAgo). LrAgo and CbAgo are DNA-guided DNA endonucleases that copurify with small DNAs upon heterologous expression in E. coli. Such guide production depends on their catalytic activity and is abolished when pAgos are rendered inactive. Small DNAs originate from both the expression plasmid and the bacterial chromosome and are enriched for plasmid-derived sequences. Well-defined guide acquisition hotspots are observed within the host chromosome that likely correspond to the preferable sites of DNA processing by pAgos. The hotspots may presumably arise at sites of frequent DNA damage and repair and do not correlate with transcription levels at corresponding regions. Our observations suggest that pAgos may sample genomic DNA in a way similar to the CRISPR adaptation apparatus. As such the DNA repair machinery may orchestrate the action of prokaryotic defence systems by facilitating nonself targeting and guide acquisition

Catalytically active Argonaute proteins from mesophilic bacteria

Argonaute proteins are an integral part of eukaryotic RNA interference machinery. They bind small noncoding RNAs and utilize them for guided cleavage of complementary RNA targets or indirect gene silencing by recruiting additional factors. Argonaute proteins are also encoded in many bacterial and archaeal genomes (pAgos). pAgos from thermophilic bacteria were initially studied to gain structural insight into eukaryotic RNA interference. They were later shown to cleave DNA substrates in a guided manner employing small RNAs or DNAs, which appear to be generated autonomously by pAgos. Thus, pAgos might be considered as means of prokaryotic defense against invasive genetic elements. Here we characterize pAgos from noncultivable or pathogenic mesophilic bacteria. Candidate proteins were selected through bioinformatic screening of genomic databases. Corresponding pAgo genes were chemically synthesized and used for expression in a heterologous system. Upon expression in E. coli these proteins have been shown to associate with short (1425 nt) 5’phosphorylated DNA molecules. Such short DNA loading relies on the catalytic activity of pAgos and is abolished in catalytically dead protein variants, which bear amino acid substitutions in the DEDX catalytic tetrad. Further in vitro assays have shown that purified pAgos cleave various DNA substrates in a guidedependent manner. They display high activity at temperatures ranging from 30 to 45 °C, with the efficiency of cleavage being greatly affected by ionic strength, supplied divalent cations and guide molecules. This suggests that all studied pAgos act as DNA dependent DNA nucleases which may subsequently be used as means of targeted genome editing in eukaryotic organisms

Functional activities of DNA-guided and RNA-guided bacterial Argonaute proteins

Specific targeting of nucleic acids by Argonaute (Ago) proteins lies at the heart of RNA interference. Eukaryotic Ago’s bind small RNAs and use them as guides for target RNA recognition and cleavage. Argonaute proteins are also found in bacterial and archaeal genomes where their roles remain unclear. Structural and biochemical studies of a few prokaryotic Ago proteins showed that they can function as endonucleases in vitro and may provide cell defense against foreign genetic elements in vivo. However, most prokaryotic Ago’s are predicted to lack endonuclease activity; they also often have unusual domain architectures and are associated in the same operons with putative nucleases or helicases. In this study, we focused on prokaryotic Ago’s from several cultivable bacterial species. We showed that although eukaryotic Ago’s work only with RNA, prokaryotic proteins included in our analysis can use either RNA or DNA guides to recognize DNA targets. To define the specificity of bacterial Ago’s we expressed them in Escherichia coli and analyzed associated short nucleic acids. Furthermore, we tested nucleolytic activity of three proteins in vitro using different guide and target molecules, and also measured the affinity of Ago’s to guides and targets. The slicer activity of Ago’s is known to depend on four conserved amino acid residues in the catalytic center. We showed that, in agreement with bioinformatic predictions, two of the three proteins possess the endonuclease activity. Our results indicate that bacterial Ago proteins can cleave target nucleic acids with high specificity in vitro and can function in the heterologous E. coli system in vivo. These properties likely underlie the process of DNA/RNA interference in bacterial cells and may promote horizontal transfer of the Ago genes

Ago nucleases from Clostridium butyricum and Limnothrix rosea can process DNA substrates at moderate temperatures

Prokaryotic Argonaute proteins (pAgos) are diverse homologs of eukaryotic Argonautes (eAgos) involved in RNA interference. In contrast to eAgos, which are RNA-guided RNA nucleases, several pAgos were reported to act as DNA nucleases suggesting that they may be used as an alternative to CRISPRC as nucleases for genome editing. However, all previously studied pAgos were isolated from thermophilic bacteria or archaea thus limiting their potential use in genomic applications. We describe two pAgo nucleases from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo). Both CbAgo and LrAgo use small DNA guides to cleave complementary DNA targets and are active at physiological temperatures. At the same time, the two proteins reveal significant variations in DNA processing depending on the reaction conditions and guide structure, including changes in the guide 5’-end and the presence of mismatches. We show that CbAgo is highly active under a wide range of conditions and can precisely cleave single-stranded and double-stranded DNA at moderate temperatures suggesting that it may be used for DNA manipulations both in vitro and in vivo

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Members of the conserved Argonaute (Ago) protein family provide defense against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases, however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with nonspecific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus, however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance

DNA interference and beyond: structure and functions of prokaryotic Argonaute proteins

Recognition and repression of RNA targets by Argonaute proteins guided by small RNAs is the essence of RNA interference in eukaryotes. Argonaute proteins with diverse structures are also found in many bacterial and archaeal genomes. Recent studies revealed that, similarly to their eukaryotic counterparts, prokaryotic Argonautes (pAgos) may function in cell defense against foreign genetic elements but, in contrast, preferably act on DNA targets. Many crucial details of the pAgo action, and the roles of a plethora of pAgos with non-conventional architecture remain unknown. Here, we review available structural and biochemical data on pAgos and discuss their possible functions in host defense and other genetic processes in prokaryotic cells