Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for ...sequence-specific recognition-and sometimes cleavage-of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are involved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing.
The eukaryotic Ago proteins and the RNA interference pathways they are involved in are widely used as a powerful tool in research and as potential therapeutics. In contrast, the properties and functions of prokaryotic Ago (pAgo) proteins have remained poorly understood. Understanding the diversity and functions of pAgos holds a huge potential for discovery of new cellular pathways and novel tools for genome manipulations. Only few pAgos have been characterized by structural or biochemical approaches, while previous genomic studies discovered about 300 proteins in archaeal and eubacterial genomes. Since that time the number of bacterial strains with sequenced genomes has greatly expanded, and many previously sequenced genomes have been revised. We undertook comprehensive analysis of pAgo proteins in sequenced genomes and almost tripled the number of known genes of this family. Our research thus forms a foundation for further experimental characterization of pAgo functions that will be important for understanding of the basic biology of these proteins and their adoption as a potential tool for genome engineering in the future.
The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA-mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses ...TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3' untranslated region of actively transcribed genes induces piRNA production towards the 3'-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.
MicroRNAs (miRNA) are short 21-23nt RNAs capable of inhibiting translation of complementary target messenger RNAs. Almost half of D. melanogaster miRNA genes are grouped in genomic clusters.
The ...peculiarities of the expression of clustered miRNAs were studied using publicly available libraries of sequenced small RNAs from different Drosophila tissues. We have shown that although miRNAs from almost all clusters have similar tissue expression profiles (coordinated clusters), some clusters contain miRNAs with uncoordinated expression profiles. The predicted transcription start sites (TSSs) of such clusters are located upstream of the first miRNA, but no TSSs are found within the clusters. The expression profiles of miR and miR* sequences in uncoordinated clustered miRNAs do not correlate while their profiles from the coordinated clustered miRNAs are similar.
The presence of exclusively upstream promoters in miRNA clusters containing uncoordinated miRNAs means that the clusters are transcribed as single transcription units. The difference of tissue expression profiles of uncoordinated miRNAs and the corresponding miRs* suggests a post-transcriptional regulation of their processing or stability.
Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA ...targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.
Abstract
The nascent polypeptide-associated complex (NAC) consisting of α- and β-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed ...co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. Here we report on discovering the germline-specific NACαβ paralogs (gNACs), whose β-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single
αNAC
gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and β subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-β subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. We revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-β sequences of
Drosophila
species are positionally conserved despite these disordered regions are drastically different. We propose the IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells. Our findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and β-subunits based on assessing their depletion effects on the fly viability and gonad development.
PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized ...heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.
miRNA‐mediated gene repression and ubiquitin‐dependent processes are among the oldest and most versatile mechanisms that control multiple molecular pathways, rather than just protein turnover. These ...systems were discovered decades ago and have become among the most studied. All systems within cells are interconnected, and these two are no exception: the plethora of studies have demonstrated that the activity of the miRNAs system depends on players of the ubiquitin‐centered universe of processes, and vice versa. This review focuses on recent progress that highlights that very similar mechanisms of regulation of miRNAs by ubiquitin‐related processes are likely to be found in distantly related species, including animals, plants, and viruses. Most of them occur through the ubiquitination of Argonaute proteins, but some of the other miRNA system factors are also regulated. This suggests that their regulatory relationships are either ancient evolutionary acquisitions or have arisen independently in different kingdoms.
In this review, we examine how proteins from the universe of ubiquitin‐driven processes regulate the miRNA system in animals, plants, and viruses. Most reviewed mechanisms tune the activity of the key factor of the miRNA system ‐ Argonaute protein—although some other factors are regulated as well.
Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must ...scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.
The testis specific X-linked genes whose evolution is traced here in the melanogaster species subgroup are thought to undergo fast rate of diversification. The CK2ßtes and NACβtes gene families ...encode the diverged regulatory β-subunits of protein kinase CK2 and the homologs of β-subunit of nascent peptide associated complex, respectively. We annotated the CK2βtes-like genes related to CK2ßtes family in the D. simulans and D. sechellia genomes. The ancestor CK2βtes-like genes preserved in D. simulans and D. sechellia are considered to be intermediates in the emergence of the D. melanogaster specific Stellate genes related to the CK2ßtes family. The CK2ßtes-like genes are more similar to the unique autosomal CK2ßtes gene than to Stellates, taking into account their peculiarities of polymorphism. The formation of a variant the CK2ßtes gene Stellate in D. melanogaster as a result of illegitimate recombination between a NACßtes promoter and a distinct polymorphic variant of CK2ßtes-like ancestor copy was traced. We found a close nonrandom proximity between the dispersed defective copies of DINE-1 transposons, the members of Helitron family, and the CK2βtes and NACβtes genes, suggesting an involvement of DINE-1 elements in duplication and amplification of these genes.
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