Effective gene silencing in Drosophila ovarian germline by artificial microRNAs.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang H
PROVIDER: S-EPMC3203660 | biostudies-literature | 2011 Apr
REPOSITORIES: biostudies-literature
Publications
Effective gene silencing in Drosophila ovarian germline by artificial microRNAs.
Wang Hailong H Mu YanJun Y Chen Dahua D
Cell research 20110322 4
Similar Datasets
Project description:MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called "two-hit" amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional "one-hit" amiRNAs. The authors also demonstrated the effectiveness of "two-hit" amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, "two-hit" amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare "two-hit" amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.
| S-EPMC10440985 | biostudies-literature
Project description:Artificial microRNAs (amiRNAs) are highly specific, 21-nucleotide (nt) small RNAs designed to silence target transcripts. In plants, their application as biotechnological tools for functional genomics or crop improvement is limited by the need of transgenically expressing long primary miRNA (pri-miRNA) precursors to produce the amiRNAs in vivo. Here, we analyzed the minimal structural and sequence requirements for producing effective amiRNAs from the widely used, 521-nt long AtMIR390a pri-miRNA from Arabidopsis thaliana. We functionally screened in Nicotiana benthamiana a large collection of constructs transiently expressing amiRNAs against endogenous genes and from artificially shortened MIR390-based precursors and concluded that highly effective and accurately processed amiRNAs can be produced from a chimeric precursor of only 89 nt. This minimal precursor was further validated in A. thaliana transgenic plants expressing amiRNAs against endogenous genes. Remarkably, minimal but not full-length precursors produce authentic amiRNAs and induce widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied into leaves by spraying infectious crude extracts. Our results reveal that the length of amiRNA precursors can be shortened without affecting silencing efficacy, and that viral vectors including minimal amiRNA precursors can be applied in a transgene-free manner to induce whole-plant gene silencing.
| S-EPMC10602918 | biostudies-literature
Project description:Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing.
| S-EPMC1462600 | biostudies-other
Project description:Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5'-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.
| S-EPMC4464980 | biostudies-literature
Project description:The study of P transposable element repression in Drosophila melanogaster led to the discovery of the trans-silencing effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences) represses in trans, in the female germline, a homologous P-lacZ transgene inserted in euchromatin. TSE shows variegation in ovaries and displays a maternal effect as well as epigenetic transmission through meiosis. In addition, TSE is highly sensitive to mutations affecting heterochromatin components (including HP1) and the Piwi-interacting RNA silencing pathway (piRNA), a homology-dependent silencing mechanism that functions in the germline. TSE appears thus to involve the piRNA-based silencing proposed to play a major role in P repression. Under this hypothesis, TSE may also be established when homology between the telomeric and target loci involves sequences other than P elements, including sequences exogenous to the D. melanogaster genome. We have tested whether TSE can be induced via lacZ sequence homology. We generated a piggyBac-otu-lacZ transgene in which lacZ is under the control of the germline ovarian tumor promoter, resulting in strong expression in nurse cells and the oocyte. We show that all piggyBac-otu-lacZ transgene insertions are strongly repressed by maternally inherited telomeric P-lacZ transgenes. This repression shows variegation between egg chambers when it is incomplete and presents a maternal effect, two of the signatures of TSE. Finally, this repression is sensitive to mutations affecting aubergine, a key player of the piRNA pathway. These data show that TSE can occur when silencer and target loci share solely a sequence exogenous to the D. melanogaster genome. This functionally supports the hypothesis that TSE represents a general repression mechanism which can be co-opted by new transposable elements to regulate their activity after a transfer to the D. melanogaster genome.
| S-EPMC3291502 | biostudies-literature
Project description:Nuclear hormone receptors have emerged as important regulators of mammalian and Drosophila adult physiology, affecting such seemingly diverse processes as adipogenesis, carbohydrate metabolism, circadian rhythm, stem cell function, and gamete production. Although nuclear hormone receptors Ecdysone Receptor (EcR) and Ultraspiracle (Usp) have multiple known roles in Drosophila development and regulate key processes during oogenesis, the adult function of the majority of nuclear hormone receptors remains largely undescribed. Ecdysone-induced protein 78C (E78), a nuclear hormone receptor closely related to Drosophila E75 and to mammalian Rev-Erb and Peroxisome Proliferator Activated Receptors, was originally identified as an early ecdysone target; however, it has remained unclear whether E78 significantly contributes to adult physiology or reproductive function. To further explore the biological function of E78 in oogenesis, we used available E78 reporters and created a new E78 loss-of-function allele. We found that E78 is expressed throughout the germline during oogenesis, and is important for proper egg production and for the maternal control of early embryogenesis. We showed that E78 is required during development to establish the somatic germline stem cell (GSC) niche, and that E78 function in the germline promotes the survival of developing follicles. Consistent with its initial discovery as an ecdysone-induced target, we also found significant genetic interactions between E78 and components of the ecdysone-signaling pathway. Taken together with the previously described roles of EcR, Usp, and E75, our results suggest that nuclear hormone receptors are critical for the broad transcriptional control of a wide variety of cellular processes during oogenesis.
| S-EPMC4448935 | biostudies-literature
Project description:Silencing of genomic repeats, including transposable elements, in Drosophila melanogaster is mediated by repeat-associated short interfering RNAs (rasiRNAs) interacting with proteins of the Piwi subfamily. rasiRNA-based silencing is thought to be mechanistically distinct from both the RNA interference and microRNA pathways. We show that the amount of rasiRNAs of a wide range of retroelements is drastically reduced in ovaries and testes of flies carrying a mutation in the spn-E gene. To address the mechanism of rasiRNA-dependent silencing of retrotransposons, we monitored their chromatin state in ovaries and somatic tissues. This revealed that the spn-E mutation causes chromatin opening of retroelements in ovaries, resulting in an increase in histone H3 K4 dimethylation and a decrease in histone H3 K9 di/trimethylation. The strongest chromatin changes have been detected for telomeric HeT-A elements that correlates with the most dramatic increase of their transcript level, compared to other mobile elements. The spn-E mutation also causes depletion of HP1 content in the chromatin of transposable elements, especially along HeT-A arrays. We also show that mutations in the genes controlling the rasiRNA pathway cause no derepression of the same retrotransposons in somatic tissues. Our results provide evidence that germinal Piwi-associated short RNAs induce chromatin modifications of their targets.
| S-EPMC2018648 | biostudies-literature
Project description:In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the "ping-pong" amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.
| S-EPMC5295597 | biostudies-literature
Project description:Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.
| S-EPMC3102283 | biostudies-literature
Project description:The maintenance of stem cell populations and the differentiation of their progeny is coordinated by specific communication with associated niche cells. Here, we describe a protocol for short-term live imaging of the Drosophila ovarian germline stem cell niche ex vivo. By immobilizing the ovarian tissue in a fibrinogen-thrombin clot, we are able to maintain the tissue for short-term high-temporal live imaging. This enables the visualization of dynamic cellular processes, such as the cytoskeletal dynamics that control stem cell niche communication. For complete details on the use and execution of this protocol, please refer to Wilcockson and Ashe (2019).
| S-EPMC7941016 | biostudies-literature