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

To be or not to be a piRNA: genomic origin and processing of piRNAs

Piwi-interacting RNAs (piRNAs) originate from genomic regions dubbed piRNA clusters. How cluster transcripts are selected for processing into piRNAs is not understood. We discuss evidence for the involvement of chromatin structure and maternally inherited piRNAs in determining their fate

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

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

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

Prokaryotic Argonautes defend genomes against invasive DNA

Argonaute proteins are central players in small RNAmediated silencing mechanisms such as RNA interference (RNAi), microRNA repression and piRNA-mediated transposon silencing. In eukaryotes, Argonautes bind small RNAs that guide them to RNA targets in order to regulate gene expression and repress invasive genomic elements. Although Argonaute proteins are conserved in all life forms from bacteria to eukaryotes, until now studies have focused on the biological functions of eukaryotic Argonautes. Here we highlight two recent studies that discover the functions of prokaryotic Argonautes in defence against exogenous DNA

An improved protocol for small RNA library construction using High Definition adapters

Next generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with dramatically reduced adapter-adapter product. This protocol was developed from the procedure invented by Vaidyanathan et al. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue. The PCR products contained a very small amount of adapter-adapter product. Bioinformatic analysis of the sequencing data revealed sRNAs with diverse sequences and many different miRNA families