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.

• MeSH
• algoritmy MeSH
• Argonaut proteiny genetika   metabolismus   MeSH
• deep learning * MeSH
• mikro RNA * genetika   metabolismus   MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Germline genome defense evolves to recognize and suppress retrotransposons. One of defensive mechanisms is the PIWI-associated RNA (piRNA) pathway, which employs small RNAs for sequence-specific repression. The loss of the piRNA pathway in mice causes male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes posses also RNA interference (RNAi), another small RNA pathway capable of retrotransposon suppression. To examine whether RNAi compensates the loss of the piRNA pathway, we produced a new RNAi pathway mutant DicerSOM and crossed it with a catalytically-dead mutant of Mili, an essential piRNA gene. Normal follicular and oocyte development in double mutants showed that RNAi does not suppress a strong ovarian piRNA knock-out phenotype. However, we observed redundant and non-redundant targeting of specific retrotransposon families illustrating stochasticity of recognition and targeting of invading retrotransposons. Intracisternal A Particle retrotransposon was mainly targeted by the piRNA pathway, MaLR and RLTR10 retrotransposons were targeted mainly by RNAi. Double mutants showed accumulations of LINE-1 retrotransposon transcripts. However, we did not find strong evidence for transcriptional activation and mobilization of retrotransposition competent LINE-1 elements suggesting that while both defense pathways are simultaneously expendable for ovarian oocyte development, yet another transcriptional silencing mechanism prevents mobilization of LINE-1 elements.

• MeSH
• Argonaut proteiny genetika   MeSH
• DEAD-box RNA-helikasy genetika   MeSH
• malá interferující RNA genetika   MeSH
• mutace MeSH
• myši MeSH
• oocyty chemie   růst a vývoj   MeSH
• retroelementy * MeSH
• ribonukleasa III genetika   MeSH
• RNA interference * MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

PIWI-interacting RNAs (piRNAs) and their associated PIWI clade Argonaute proteins constitute the core of the piRNA pathway. In gonadal cells, this conserved pathway is crucial for genome defense, and its main function is to silence transposable elements. This is achieved through posttranscriptional and transcriptional gene silencing. Precursors that give rise to piRNAs require specialized transcription and transport machineries because piRNA biogenesis is a cytoplasmic process. The ping-pong cycle, a posttranscriptional silencing mechanism, combines the cleavage-dependent silencing of transposon RNAs with piRNA production. PIWI proteins also function in the nucleus, where they scan for nascent target transcripts with sequence complementarity, instructing transcriptional silencing and deposition of repressive chromatin marks at transposon loci. Although studies have revealed numerous factors that participate in each branch of the piRNA pathway, the precise molecular roles of these factors often remain unclear. In this review, we summarize our current understanding of the mechanisms involved in piRNA biogenesis and function.

• MeSH
• Argonaut proteiny genetika   MeSH
• Drosophila melanogaster genetika   MeSH
• genetická transkripce * MeSH
• gonády růst a vývoj   MeSH
• malá interferující RNA biosyntéza   genetika   MeSH
• transpozibilní elementy DNA genetika   MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

• MeSH
• Argonaut proteiny fyziologie   MeSH
• buněčné linie MeSH
• klíště genetika   virologie   MeSH
• malá interferující RNA chemie   MeSH
• malá nekódující RNA chemie   MeSH
• ribonukleasa III fyziologie   MeSH
• RNA interference * MeSH
• RNA virová chemie   MeSH
• viry klíšťové encefalitidy genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural 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