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Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements.

Makarova KS, Wolf YI, van der Oost J, Koonin EV - Biol. Direct (2009)

Bottom Line: Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases.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.The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Center for Biotechnology Information, NLM, National Institutes of Health, Bethesda, Maryland 20894, USA. makarova@ncbi.nlm.nih.gov

ABSTRACT

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. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown.

Results: We present a 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 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 associated with genes for 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 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.

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Phylogenetic analysis of PIWI-domains and organization of the predicted pAgo operons. The ML tree is rooted between the (predominantly) PAZ-domain-containing and PAZ-domain- lacking branches. The RELL bootstrap values are indicated (%) for selected major branches. Color code: gray, Eukaryota; orange, Archaea; blue, Proteobacteria, green, Firmicutes; black, other lineages of bacteria. Each organism is denoted by the full systematic name and the Gene Identifier (GI) number. The PDB ID is indicated for those sequences for which tertiary structure is solved. Sequences of short PIWI proteins (that have lost N-terminal part including PAZ domain) but belong to the branch that consists mostly of full size sequences are indicated by "#" symbol. For those PIWI-domain proteins that are associated with genes encoding a nuclease domain, the domain architectures of the pAgo-associated proteins are shown.
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Figure 3: Phylogenetic analysis of PIWI-domains and organization of the predicted pAgo operons. The ML tree is rooted between the (predominantly) PAZ-domain-containing and PAZ-domain- lacking branches. The RELL bootstrap values are indicated (%) for selected major branches. Color code: gray, Eukaryota; orange, Archaea; blue, Proteobacteria, green, Firmicutes; black, other lineages of bacteria. Each organism is denoted by the full systematic name and the Gene Identifier (GI) number. The PDB ID is indicated for those sequences for which tertiary structure is solved. Sequences of short PIWI proteins (that have lost N-terminal part including PAZ domain) but belong to the branch that consists mostly of full size sequences are indicated by "#" symbol. For those PIWI-domain proteins that are associated with genes encoding a nuclease domain, the domain architectures of the pAgo-associated proteins are shown.

Mentions: We constructed a phylogenetic tree of the PIWI domains from all the detected pAgos (after excluding sequences that were fragmented or truncated due to poor annotation) and a subset of eukaryotic Argonautes (Figure 3). The majority of the PIWI domains from pAgos that lack a PAZ domain form a distinct clade although a few of these short forms cluster within the other clade that consists mostly of full-size, PAZ-containing pAgos. Within the latter clade, the short proteins do not form a distinct group (Figure 3), suggesting the N-terminal part of pAgo was lost independently in several lineages. Consistent with the similarity of domain architectures and with the results of previous analyses [29], eukaryotic Argonautes belong to a well-supported clade together with a distinct subset of archaeal pAgos; in particular the structurally characterized Pyrococcus furiosus protein, that is considered to be the model for Argonaute functioning in eukaryotes [33]. Other archaeal proteins are scattered in the tree, suggesting multiple horizontal gene transfers (HGT) between bacteria and archaea (Figure 3). Despite the existence of several small lineage-specific groups (alpha proteobacteria, gamma proteobacteria, bacteroides and cyanobacteria), the results of our phylogenetic analysis strongly suggest that pAgo genes mostly disseminated by HGT; the patchy distribution of these genes makes it unlikely that they perform indispensible functions in any bacteria or archaea (Figure 3).


Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements.

Makarova KS, Wolf YI, van der Oost J, Koonin EV - Biol. Direct (2009)

Phylogenetic analysis of PIWI-domains and organization of the predicted pAgo operons. The ML tree is rooted between the (predominantly) PAZ-domain-containing and PAZ-domain- lacking branches. The RELL bootstrap values are indicated (%) for selected major branches. Color code: gray, Eukaryota; orange, Archaea; blue, Proteobacteria, green, Firmicutes; black, other lineages of bacteria. Each organism is denoted by the full systematic name and the Gene Identifier (GI) number. The PDB ID is indicated for those sequences for which tertiary structure is solved. Sequences of short PIWI proteins (that have lost N-terminal part including PAZ domain) but belong to the branch that consists mostly of full size sequences are indicated by "#" symbol. For those PIWI-domain proteins that are associated with genes encoding a nuclease domain, the domain architectures of the pAgo-associated proteins are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743648&req=5

Figure 3: Phylogenetic analysis of PIWI-domains and organization of the predicted pAgo operons. The ML tree is rooted between the (predominantly) PAZ-domain-containing and PAZ-domain- lacking branches. The RELL bootstrap values are indicated (%) for selected major branches. Color code: gray, Eukaryota; orange, Archaea; blue, Proteobacteria, green, Firmicutes; black, other lineages of bacteria. Each organism is denoted by the full systematic name and the Gene Identifier (GI) number. The PDB ID is indicated for those sequences for which tertiary structure is solved. Sequences of short PIWI proteins (that have lost N-terminal part including PAZ domain) but belong to the branch that consists mostly of full size sequences are indicated by "#" symbol. For those PIWI-domain proteins that are associated with genes encoding a nuclease domain, the domain architectures of the pAgo-associated proteins are shown.
Mentions: We constructed a phylogenetic tree of the PIWI domains from all the detected pAgos (after excluding sequences that were fragmented or truncated due to poor annotation) and a subset of eukaryotic Argonautes (Figure 3). The majority of the PIWI domains from pAgos that lack a PAZ domain form a distinct clade although a few of these short forms cluster within the other clade that consists mostly of full-size, PAZ-containing pAgos. Within the latter clade, the short proteins do not form a distinct group (Figure 3), suggesting the N-terminal part of pAgo was lost independently in several lineages. Consistent with the similarity of domain architectures and with the results of previous analyses [29], eukaryotic Argonautes belong to a well-supported clade together with a distinct subset of archaeal pAgos; in particular the structurally characterized Pyrococcus furiosus protein, that is considered to be the model for Argonaute functioning in eukaryotes [33]. Other archaeal proteins are scattered in the tree, suggesting multiple horizontal gene transfers (HGT) between bacteria and archaea (Figure 3). Despite the existence of several small lineage-specific groups (alpha proteobacteria, gamma proteobacteria, bacteroides and cyanobacteria), the results of our phylogenetic analysis strongly suggest that pAgo genes mostly disseminated by HGT; the patchy distribution of these genes makes it unlikely that they perform indispensible functions in any bacteria or archaea (Figure 3).

Bottom Line: Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases.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.The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Center for Biotechnology Information, NLM, National Institutes of Health, Bethesda, Maryland 20894, USA. makarova@ncbi.nlm.nih.gov

ABSTRACT

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. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown.

Results: We present a 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 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 associated with genes for 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 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.

Show MeSH
Related in: MedlinePlus