Biology subjects

Ryazansky, S.

Publications and source records attributed to Ryazansky, S..

7 recordsLinked to original sources

bioRxiv2025

Dora, a key component of target-directed miRNA degradation, is essential for local genomic amplification in Drosophila ovarian follicle cells

The ubiquitin ligase receptor Dora, the Drosophila homolog of ZSWIM8, is a key component of the target-directed microRNA degradation (TDMD) pathway. Previous studies have implicated TDMD - and, consequently, ZSWIM8/Dora - in various developmental processes. Here, we investigate the role of Dora in Drosophila oogenesis, focusing on its function in ovarian somatic cells. We generated a fly strain with an endogenously tagged Dora protein and observed its presence in both germline and somatic follicular cells of the ovaries. Somatic knockdown of dora revealed its essential role in normal eggshell formation. Specifically, its loss led to reduced chorion and vitelline transcript levels and decreased chorion gene amplification, both of which are critical for eggshell protein production. Somatic depletion of Dora did not affect the abundance of other known regulators of eggshell formation, including Ttk69, Cut, miR-7 or miR-318 indicating that Dora functions independently of these pathways. Although a direct link between TDMD and chorion eggshell development remains to be confirmed, our findings clearly highlight Dora as a critical regulator in this process. These results pave the way for further investigation into the specific role of TDMD and provide new insights into the regulatory mechanisms underlying animal oogenesis.

developmental biology↗

bioRxiv2023

Evidence of target-mediated miRNA degradation in Drosophila ovarian cell culture

Target-mediated miRNA degradation (TDMD) is a recently discovered process of post-transcriptional regulation of miRNA stability in animals. TDMD is induced by the formation of the non-canonical duplex of Ago-bound miRNAs with the specialized RNA target, and, as suggested for human cell culture, this complex is recognized by the ZSWIM8 receptor protein of the Cullin-RING-ligase complex CRL3. CRL3 ubiquitinates Ago, resulting in proteolysis of Ago and degradation of the released miRNAs. To date, the molecular mechanism of the TDMD process was not studied in other animal species. Here we investigated protein Dora, the Drosophila ortholog of ZSWIM8, in the culture of Drosophila ovarian somatic cells (OSC). We show that Dora in OSCs localizes in protein granules that are not related to P-and GW-bodies. The knock-out of Dora up-regulates multiple miRNAs, including miR-7-5p. Also, we show that Dora associates with proteins of the CRL3 complex, and the depletion of its main component Cul3 up-regulates miR-7-5p. We concluded that the mechanism of TDMD is conserved in humans and Drosophila. The knock-out of Dora also down-regulates the putative protein-coding targets of miRNAs. One of them is Tom from the Brd-C gene family, which is known to repress the Notch signaling pathway. Indeed, in cells lacking Dora, we have observed the down-regulation of cut, the marker of the activated Notch pathway. This data indicates that TDMD in OSCs may contribute to modulation of the Notch pathway.

molecular biology↗

bioRxiv2022

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) that were similarly proposed to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and very little is known about their functional activity and target specificity in vivo. Here, we describe five pAgo proteins from mesophilic bacteria that act as DNA-guided DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmid DNA and from chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum can induce DNA interference between multicopy sequences resulting in targeted processing of homologous plasmid and chromosomal loci. EmaAgo also protects bacteria from bacteriophage infection and is preferentially loaded with phage guide DNAs suggesting that the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

molecular biology↗

bioRxiv2020

pAgo-induced DNA interference protects bacteria from invader DNA

Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species 1-3. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference 4-8. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.

molecular biology↗

bioRxiv2019

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

Argonaute (Ago) proteins are the key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleave of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In contrast to previously studied pAgos, CbAgo and LrAgo can use not only 5-phosphorylated but also 5-hydroxyl DNA guides, with diminished precision of target cleavage. Both LrAgo and CbAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3-guide region. CbAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both single-stranded and double-stranded DNA substrates at moderate temperatures. The biochemical characterization of mesophilic pAgo proteins paths the way for their use for DNA manipulations both in vitro and in vivo.

molecular biology↗

bioRxiv2018

The expanded universe of prokaryotic Argonaute proteins

The 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 almost tripled the number of previously analyzed genes of this family. In comparison with eAgos, pAgos are highly diverse and have likely spread by horizontal gene transfer. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids and in target cleavage, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and processing. Many pAgos, including catalytically inactive variants, are associated with putative nucleases, helicases and DNA binding proteins in the same gene or operon, suggesting that they are involved in DNA processing. The great diversity of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and their use as potential tools in genome editing.

genomics↗

bioRxiv2017

Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline

Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) were discovered in different species, however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we show that the piRNA pathway provides a strong germline-specific mechanism of telomere homeostasis. We show that telomeric retrotransposon arrays belong to a unique class of dual-strand piRNA clusters whose transcripts, required for telomere elongation, serve simultaneously as piRNA precursors and their only targets. However, the ability to produce piRNAs and bind Rhino - a germline-specific homolog of heterochromatic protein 1 (HP1) - varies along telomeres. Most likely, this heterogeneity is determined by the peculiarities of telomeric retrotransposons themselves. piRNAs play a pivotal role in the establishment and maintenance of telomeric and subtelomeric chromatin in the germline facilitating loading of HP1 and histone 3 lysine 9 trimethylation mark - highly conservative telomere components - at different telomeric regions. piRNA pathway disruption results in telomere dysfunction characterized by a loss of heterochromatic components and translocation of telomeres from the periphery to the nuclear interior but does not affect the telomere end capping.

genomics↗