Bacterial Argonaute Samples the Transcriptome to Identify Foreign DNA

Division of Biology, California Institute of Technology, 147-75, 1200E California Boulevard, Pasadena, CA 91125, USA
Molecular cell (Impact Factor: 14.02). 09/2013; 51(5):594-605. DOI: 10.1016/j.molcel.2013.08.014
Source: PubMed


Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea, their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid-derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids.

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Available from: Ivan Olovnikov
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    • "In particular, multiple proviruses have been identified in archaea, and it has been shown that not only tailed bacteriophages, well known to integrate into bacterial chromosomes, but also ssDNA viruses form numerous proviruses (Krupovic and Forterre 2011). In parallel, in recent years, fueled in large by the discovery of the CRISPR-Cas systems of archaeal and bacterial adaptive immunity and their application in genome engineering (Barrangou and Horvath 2012; Kim and Kim 2014; Makarova et al. 2013a, b; Marraffini and Sontheimer 2010), comparative genomic and experimental studies have revealed an unsuspected diversity of prokaryotic defense systems that function on the principles of innate immunity, adaptive immunity or programmed cell death (Makarova et al. 2013a, b; Olovnikov et al. 2013; Swarts et al. 2014). Furthermore, it has been shown that analogous to previously characterized pathogenicity and symbiosis islands, genes encoding components of defense systems tend to form cluster in bacterial and archaeal genomes. "
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    ABSTRACT: Microbial genomes encompass a sizable fraction of poorly characterized, narrowly spread fast-evolving genes. Using sensitive methods for sequences comparison and protein structure prediction, we performed a detailed comparative analysis of clusters of such genes, which we denote “dark matter islands”, in archaeal genomes. The dark matter islands comprise up to 20 % of archaeal genomes and show remarkable heterogeneity and diversity. Nevertheless, three classes of entities are common in these genomic loci: (a) integrated viral genomes and other mobile elements; (b) defense systems, and (c) secretory and other membrane-associated systems. The dark matter islands in the genome of thermophiles and mesophiles show similar general trends of gene content, but thermophiles are substantially enriched in predicted membrane proteins whereas mesophiles have a greater proportion of recognizable mobile elements. Based on this analysis, we predict the existence of several novel groups of viruses and mobile elements, previously unnoticed variants of CRISPR-Cas immune systems, and new secretory systems that might be involved in stress response, intermicrobial conflicts and biogenesis of novel, uncharacterized membrane structures. Electronic supplementary material The online version of this article (doi:10.1007/s00792-014-0672-7) contains supplementary material, which is available to authorized users.
    Full-text · Article · Aug 2014 · Extremophiles
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    • "A recent study analyzing Argonaute-associated DNA/RNA molecules in Rhodobacter sphaeroides (Rs) identified both DNA and RNA molecules that are associated with the Argonaute protein, RsAgo (Olovnikov et al., 2013). Fifteen to 19 nt RNA and 22–24 nt DNA molecules were recovered from purified Argonaute complexes (Figure 2). "
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    ABSTRACT: A general feature of Argonaute-dependent small RNAs is their base-paired precursor structures, and precursor duplex structures are often required for confident annotation of miRNA genes. However, this rule has been broken by discoveries of functional small RNA species whose precursors lack a predictable double-stranded (ds-) RNA structure, arguing that duplex structures are not prerequisite for small RNA loading to Argonautes. The biological significance of single-stranded (ss-) RNA loading has been recognized particularly in systems where active small RNA amplification mechanisms are involved, because even a small amount of RNA molecules can trigger the production of abundant RNA species leading to profound biological effects. However, even in the absence of small RNA amplification mechanisms, recent studies have demonstrated that potent gene silencing can be achieved using chemically modified synthetic ssRNAs that are resistant to RNases in mice. Therefore, such ssRNA-mediated gene regulation may have broader roles than previously recognized, and the findings have opened the door for further research to optimize the design of ss-siRNAs toward future pharmaceutical and biomedical applications of gene silencing technologies. In this review, we will summarize studies about endogenous ssRNA species that are bound by Argonaute proteins and how ssRNA precursors are recognized by various small RNA pathways.
    Full-text · Article · Jun 2014 · Frontiers in Genetics
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    • "Indeed, REase domains which are present in several CARF proteins typically targeting alien DNA whereas self DNA is targeted only under exceptional circumstances. The REases achieve this selectivity by either targeting DNA with specific modified nucleotides, such as hydroxymethylcytosine (e.g., Mrr, McrA, and McrB systems) (Bickle and Kruger, 1993; Burroughs et al., 2013b), or by targeting unmodified DNA in contrast to the host DNA that is methylated by cognate methylases (Roberts et al., 2007), and probably also by using RNA or DNA guides supplied by Argonaute (PIWI) family proteins (Makarova et al., 2009; Burroughs et al., 2013a,b; Olovnikov et al., 2013). Thus, we propose that CARF proteins containing C-terminal REase domains function in parallel with the Cascade-like complexes resulting in a double-pronged assault on the invading DNA. "
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    ABSTRACT: CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold). Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and "effector" domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.
    Full-text · Article · Apr 2014 · Frontiers in Genetics
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