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

Non-coding RNAs in Transcriptional Regulation

Transcriptional gene silencing guided by small RNAs is a process conserved from protozoa to mammals. Small RNAs loaded into Argonaute family proteins direct repressive histone modifications or DNA cytosine methylation to homologous regions of the genome. Small RNA-mediated transcriptional silencing is required for many biological processes, including repression of transposable elements, maintaining the genome stability/integrity, and epigenetic inheritance of gene expression. Here, we will summarize the current knowledge about small RNA biogenesis and mechanisms of transcriptional regulation in plants, Drosophila, Caenorhabditis elegans, and mice. Furthermore, a rapidly growing number of long non-coding RNAs (lncRNAs) have been implicated as important players in transcription regulation. We will discuss current models for long non-coding RNA-mediated gene regulation

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

RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility

piRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. In Drosophila female germline, many piRNA source loci dubbed "piRNA clusters" lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. It remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linked Suppressor of Stellate piRNAs and therefore Stellate silencing. Despite intact Stellate repression, rhi mutant males exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyond Stellate silencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus, AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis

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

piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments

Small RNA in the nucleus: the RNA-chromatin ping-pong

Eukaryotes use several classes of small RNA molecules to guide diverse protein machineries to target messenger RNA. The role of small RNA in post-transcriptional regulation of mRNA stability and translation is now well established. Small RNAs can also guide sequence-specific modification of chromatin structure and thus contribute to establishment and maintenance of distinct chromatin domains. In this review we summarize the model for the inter-dependent interaction between small RNA and chromatin that has emerged from studies on fission yeast and plants. We focus on recent results that link a distinct class of small RNAs, the piRNAs, to chromatin regulation in animals