In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Moreover, AGO fPAR-CLIP in Lamin A KO SHSY5Y cells revealed significantly reduced RNAi activity. Further exploration of the nuclear AGO interactome by mass spectrometry identified FAM120A, an RNA-binding protein and known interactor of AGO2. Subsequent FAM120A fPAR-CLIP, revealed that FAM120A co-binds AGO targets and that this competition reduces the RNAi activity. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, FAM120A mediated RNAi impairment, and upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.
- MeSH
- aktivní transport - buněčné jádro MeSH
- Argonaut proteiny * metabolismus genetika MeSH
- buněčné jádro * metabolismus MeSH
- lamin typ A * metabolismus genetika MeSH
- lidé MeSH
- melanom genetika metabolismus patologie MeSH
- mikro RNA * metabolismus genetika MeSH
- nádorové buněčné linie MeSH
- proliferace buněk * genetika MeSH
- proteiny vázající RNA metabolismus genetika MeSH
- RNA interference * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The binding of microRNAs (miRNAs) to their target sites is a complex process, mediated by the Argonaute (Ago) family of proteins. The prediction of miRNA:target site binding is an important first step for any miRNA target prediction algorithm. To date, the potential for miRNA:target site binding is evaluated using either co-folding free energy measures or heuristic approaches, based on the identification of binding 'seeds', i.e., continuous stretches of binding corresponding to specific parts of the miRNA. The limitations of both these families of methods have produced generations of miRNA target prediction algorithms that are primarily focused on 'canonical' seed targets, even though unbiased experimental methods have shown that only approximately half of in vivo miRNA targets are 'canonical'. Herein, we present miRBind, a deep learning method and web server that can be used to accurately predict the potential of miRNA:target site binding. We trained our method using seed-agnostic experimental data and show that our method outperforms both seed-based approaches and co-fold free energy approaches. The full code for the development of miRBind and a freely accessible web server are freely available.
Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.
- MeSH
- Argonaut proteiny chemie metabolismus MeSH
- guide RNA, Kinetoplastida chemie metabolismus MeSH
- hořčík MeSH
- hybridizace nukleových kyselin MeSH
- hydrolýza MeSH
- komplex RISC MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- malá interferující RNA chemie genetika metabolismus MeSH
- messenger RNA chemie metabolismus MeSH
- metoda Monte Carlo MeSH
- párování bází MeSH
- racionální návrh léčiv MeSH
- RNA interference * MeSH
- RNA virová antagonisté a inhibitory chemie MeSH
- simulace molekulového dockingu MeSH
- termodynamika MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transposable elements (TEs) are powerful drivers of genome evolutionary dynamics but are principally deleterious to the host organism by compromising the integrity and function of the genome. The transposition of TEs may result in mutations and DNA damage. DNA double-strand breaks (DSBs), which may be caused by the transposition, are one of the processes directly linked to aging. TEs may thus be considered to constitute an internal source of aging and the frequency of transposition may, in turn, be considered to affect the pace of aging. The PIWI-piRNA pathway is a widespread strategy used by most animals to effectively suppress transposition. Interestingly, the PIWI-piRNA pathway is expressed predominantly in the animal germline, a more or less continuous immortal lineage set aside after the first few cell divisions of a developing embryo. Recent findings further imply that the PIWI-piRNA pathway and TE suppression constitute an important mechanism regulating aging. This article discusses the proposed role of the PIWI-piRNA pathway in setting the pace of aging as well as the possible mechanisms underlying this process.
- MeSH
- Argonaut proteiny * genetika metabolismus MeSH
- buněčné dělení genetika MeSH
- lidé MeSH
- malá interferující RNA * genetika metabolismus MeSH
- poškození DNA * MeSH
- stárnutí * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
B chromosomes (Bs) are supernumerary, dispensable parts of the nuclear genome, which appear in many different species of eukaryote. So far, Bs have been considered to be genetically inert elements without any functional genes. Our comparative transcriptome analysis and the detection of active RNA polymerase II (RNAPII) in the proximity of B chromatin demonstrate that the Bs of rye (Secale cereale) contribute to the transcriptome. In total, 1954 and 1218 B-derived transcripts with an open reading frame were expressed in generative and vegetative tissues, respectively. In addition to B-derived transposable element transcripts, a high percentage of short transcripts without detectable similarity to known proteins and gene fragments from A chromosomes (As) were found, suggesting an ongoing gene erosion process. In vitro analysis of the A- and B-encoded AGO4B protein variants demonstrated that both possess RNA slicer activity. These data demonstrate unambiguously the presence of a functional AGO4B gene on Bs and that these Bs carry both functional protein coding genes and pseudogene copies. Thus, B-encoded genes may provide an additional level of gene control and complexity in combination with their related A-located genes. Hence, physiological effects, associated with the presence of Bs, may partly be explained by the activity of B-located (pseudo)genes.
- MeSH
- amplifikace genu MeSH
- Argonaut proteiny metabolismus MeSH
- buněčné jádro metabolismus MeSH
- chromatin metabolismus MeSH
- chromozomy rostlin genetika MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- genetická transkripce MeSH
- genová dávka MeSH
- genová ontologie MeSH
- messenger RNA genetika metabolismus MeSH
- počítačová simulace MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné geny MeSH
- rostlinné proteiny metabolismus MeSH
- sekvence nukleotidů MeSH
- žito enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The PIWIL (P-element induced wimpy testis like protein) subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA) biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA). METHODS: The expression of PIWIL genes in RA and osteoarthritis (OA) synovial tissues and synovial fibroblasts (SF) was analysed by Real-time PCR, immunofluorescence and Western blot. The expression of piRNAs was quantified by next generation small RNA sequencing (NGS). The regulation of PIWI/piRNAs, proliferation and methylation of LINE-1 after silencing of PIWIL genes were studied. RESULTS: PIWIL2 and 4 mRNA were similarly expressed in synovial tissues and SF from RA and OA patients. However, on the protein level only PIWIL4 was strongly expressed in SF. Using NGS up to 300 piRNAs were identified in all SF without significant differences in expression levels between RA and OASF. Of interest, the analysis of the co-expression of the detected piRNAs revealed a less tightly regulated pattern of piRNA-823, -4153 and -16659 expression in RASF. In RASF and OASF, stimulation with TNFα+IL1β/TLR-ligands further significantly increased the expression levels of PIWIL2 and 4 mRNA and piRNA-16659 was significantly (4-fold) induced upon Poly(I:C) stimulation. Silencing of PIWIL2/4 neither affect LINE-1 methylation/expression nor proliferation of RASF. CONCLUSION: We detected a new class of small regulatory RNAs (piRNAs) and their specific binding partners (PIWIL2/4) in synovial fibroblasts. The differential regulation of co-expression of piRNAs in RASF and the induction of piRNA/Piwi-proteins by innate immune stimulators suggest a role in inflammatory processes.
- MeSH
- Argonaut proteiny genetika metabolismus MeSH
- cytokiny metabolismus MeSH
- dlouhé rozptýlené jaderné elementy MeSH
- fibroblasty patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- osteoartróza genetika metabolismus patologie MeSH
- proteiny genetika metabolismus MeSH
- regulace genové exprese MeSH
- revmatoidní artritida genetika metabolismus patologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- synoviální membrána patologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The PIWI-interacting RNA (piRNA) pathway is a conserved defense mechanism that protects the genetic information of animal germ cells from the deleterious effects of molecular parasites, such as transposons. Discovered nearly a decade ago, this small RNA silencing system comprises PIWI-clade Argonaute proteins and their associated RNA-binding partners, the piRNAs. In this review, we highlight recent work that has advanced our understanding of how piRNAs preserve genome integrity across generations. We discuss the mechanism of piRNA biogenesis, give an overview of common themes as well as differences in piRNA-mediated silencing between species, and end by highlighting known and emerging functions of piRNAs.
- MeSH
- Argonaut proteiny genetika metabolismus MeSH
- chromozomální proteiny, nehistonové genetika metabolismus MeSH
- Drosophila melanogaster genetika metabolismus MeSH
- genetická transkripce MeSH
- histony genetika metabolismus MeSH
- lidé MeSH
- malá interferující RNA genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- proteiny asociované s mikrotubuly genetika metabolismus MeSH
- proteiny Drosophily genetika metabolismus MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- umlčování genů * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two interrelated branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborate pathway centered on the three gonad-specific Argonaute proteins (Piwi, Aubergine, and Argonaute 3). While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals key factors of piRNA-mediated transposon silencing, including the piRNA biogenesis factors CG2183 (GASZ) and Deadlock. Our data uncover a previously unanticipated set of factors preferentially required for repression of different transposon types.
- MeSH
- Argonaut proteiny genetika metabolismus MeSH
- Drosophila melanogaster genetika MeSH
- iniciační faktory genetika metabolismus MeSH
- malá interferující RNA genetika metabolismus MeSH
- ovarium fyziologie MeSH
- proteiny asociované s mikrotubuly genetika metabolismus MeSH
- proteiny Drosophily genetika metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- RNA interference * MeSH
- stanovení celkové genové exprese metody MeSH
- transpozibilní elementy DNA * MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
