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At least 217 records · Page 12Linked to original sources
PubMed2005

Molecular mechanisms of RNAi: implications for development and disease.

Research over the past few years has led to dramatic new discoveries on the role of double-stranded RNA (dsRNA) in the cell. RNA duplexes have been shown to orchestrate epigenetic changes, repress translation, and direct mRNA degradation in a sequence-specific manner. These diverse effects of dsRNA on gene expression have been termed RNA interference (RNAi). In addition to playing a role in viral defense and silencing transposons, RNAi also has a critical function in a number of developmental processes in the embryo. In this review, we explore these roles and discuss the molecular mechanisms behind dsRNA-mediated gene silencing. Further, we address the use of RNAi as a tool to study gene function in biology, and as a strategy for treating human disease.

Animals↗
PubMed2026

De novo transcriptome meta-analysis reveals candidate genes involved in life-stage transitions for RNAi-mediated management of the citrus root weevil (Diaprepes abbreviatus).

BACKGROUND: The citrus root weevil, Diaprepes abbreviatus, is a destructive agricultural pest for which molecular control options remain limited due to historically sparse genomic resources. Leveraging a comprehensive de novo transcriptome, we investigated developmental gene regulation across larval, pupal, and adult stages and identified essential targets for RNA interference (RNAi)-based intervention. RESULTS: Stage-resolved transcriptomic analyses revealed extensive transcriptional reprogramming associated with metabolism, detoxification, cuticle biosynthesis, endocrine signaling, and sensory perception. Among these, chitin synthase (DaCHS) emerged as a critical developmental gene, exhibiting pronounced up-regulation during late larval and pupal stages corresponding to intensive cuticle synthesis. Phylogenetic and structural analyses demonstrated that DaCHS is highly conserved among insects and retains canonical catalytic domains and transmembrane topology. Alpha Fold-based structural modeling and molecular docking confirmed stable interaction of DaCHS with its substrate, N-acetylglucosamine, supporting functional conservation of enzymatic activity. Oral delivery of DaCHS double-stranded RNA induced robust transcript suppression, leading to significant mortality and severe developmental defects, including larval and pupal abnormalities, and adults with disrupted wing and abdominal morphogenesis. CONCLUSION: These findings establish DaCHS as an indispensable gene for D. abbreviates development and validate transcriptome-guided RNAi as a powerful framework for target discovery. This work provides a strong molecular foundation for developing RNAi-based strategies that can be integrated into sustainable management programs for citrus root weevil control. © 2026 Society of Chemical Industry.

Animals↗
PubMed2002

Analysis of the role of RecQ helicases in RNAi in mammals.

The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.

Adenosine Triphosphatases↗
PubMed2005

Towards automated cellular image segmentation for RNAi genome-wide screening.

The Rho family of small GTPases is essential for morphological changes during normal cell development and migration, as well as during disease states such as cancer. Our goal is to identify novel effectors of Rho proteins using a cell-based assay for Rho activity to perform genome-wide functional screens using double stranded RNA (dsRNAs) interference. We aim to discover genes could cause the cell phenotype changed dramatically. Biologists currently attempt to perform the genome-wide RNAi screening to identify various image phenotypes. RNAi genome-wide screening, however, could easily generate more than a million of images per study, manual analysis is thus prohibitive. Image analysis becomes a bottleneck in realizing high content imaging screens. We propose a two-step segmentation approach to solve this problem. First, we determine the center of a cell using the information in the DNA-channel by segmenting the DNA nuclei and the dissimilarity function is employed to attenuate the over-segmentation problem, then we estimate a rough boundary for each cell using a polygon. Second, we apply fuzzy c-means based multi-threshold segmentation and sharpening technology; for isolation of touching spots, marker-controlled watershed is employed to remove touching cells. Furthermore, Voronoi diagrams are employed to correct the segmentation errors caused by overlapping cells. Image features are extracted for each cell. K-nearest neighbor classifier (KNN) is employed to perform cell phenotype classification. Experimental results indicate that the proposed approach can be used to identify cell phenotypes of RNAi genome-wide screens.

Algorithms↗
PubMed2004

Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties.

The caffeine biosynthetic pathway in coffee plants has been proposed to involve three distinct N -methyltransferases, xanthosine methyltransferase (XMT), 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase). We previously isolated all corresponding cDNAs designated as CaXMT1 , CaMXMT1 , CaMXMT2 and CaDXMT1 , respectively, and showed that caffeine was indeed synthesized in vitro by the combination of their gene products. In order to regulate caffeine biosynthesis in planta , we suppressed expression of CaMXMT1 by the double stranded RNA interference (RNAi) method. For this purpose, we first established a protocol for efficient somatic embryogenesis of Coffea arabica and C. canephora , and then Agrobacterium -mediated transformation techniques. The RNAi transgenic lines of embryogenic tissues derived from C. arabica and transgenic plantlets of C. canephora demonstrated a clear reduction in transcripts for CaMXMT1 in comparison with the control plants. Transcripts for CaXMT1 and CaDXMT1 were also reduced in the most cases. Both embryonic tissues and plantlets exhibited a concomitant reduction of theobromine and caffeine contents to a range between 30% and 50% of that of the control. These results suggest that the CaMXMT1 -RNAi sequence affected expression of not only CaMXMT1 itself, but also CaXMT1 and CaDXMT1 , and that, since the reduction in theobromine content was proportional to that for caffeine, it is involved in the major synthetic pathway in coffee plants. The results also indicate that the method can be practically applied to produce decaffeinated coffee plants.

Caffeine↗
PubMed2004

Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells.

Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Knockdown of p53 by RNAi in ES cells facilitates RA-induced differentiation into muscle cells.

The p53 gene is widely expressed in embryo, tissues, and tumors, and its deficiency can rescue embryonic defects in certain genes null embryos. However, it is still poorly understood whether p53 is involved in myoblast and neuronal fate determination during embryogenesis. We established the ES cell clone in which p53 protein was persistently suppressed by stable expression of p53 RNAi, and GFP was expressed in a p53 RNAi transcription-independent manner. With the classical protocol in which the differentiation of ES cells into either neural or muscle cell is specifically modulated by different dosage retinoic acid (RA), we evaluated the function of p53 during myoblast and neuronal commitment. With RA treatment, silencing of p53 by RNAi in ES cells leads to dominant muscle cell production but lack of neuronal cell, indicating that p53 indeed plays a role during muscle and neuronal fate commitment. It thus provides a good model for investigating cross-talk between RA and p53 pathways during myogenesis and neurogenesis from ES cells.

Base Sequence↗
PubMed2005

RNAi as a tool to study cell biology: building the genome-phenome bridge.

In the few short years since its discovery, RNA interference (RNAi) has revolutionized the functional analysis of genomes: both technical and conceptual approaches to the investigation of gene function are being transformed as a result of this new technology. Genome-scale RNAi analyses have already been performed in the model organisms Caenorhabditis elegans (in vivo) and Drosophila melanogaster (in cell lines), ushering in a new era of RNAi-based approaches to probing the inner workings of the cell. The transformation of complex phenotypic data into mineable 'digitized' formats is fostering the emergence of a new area of bioinformatics related to the phenome.

Animals↗
PubMed2006

RNAi components are required for nuclear clustering of Polycomb group response elements.

Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. We show here that components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets.

Animals↗
PubMed2006

A long-distance relationship between RNAi and Polycomb.

RNA interference (RNAi) pathways can result in sequence-specific transcriptional gene silencing on the level of chromatin. In this issue of Cell, Grimaud et al. (2006) reveal that the RNAi machinery is required for long-distance physical interactions between chromosomes mediated by the Polycomb repressive complex. These results suggest that the RNAi machinery may regulate higher-order nuclear organization.

Cell Nucleus↗
PubMed2005

Uncovering RNAi mechanisms in plants: biochemistry enters the foray.

In plants, the RNA interference (RNAi) machinery responds to a variety of triggers including viral infection, transgenes, repeated elements and transposons. All of these triggers lead to silencing outcomes ranging from mRNA degradation to translational repression to chromatin remodeling. Thus, plants offer us a potentially unique opportunity to understand the full range of RNAi effector mechanisms. In this review, we discuss the recent developments in our understanding of plant RNAi mechanisms from a biochemical perspective.

Animals↗
PubMed2005

Small RNA asymmetry in RNAi: function in RISC assembly and gene regulation.

RNAi is a conserved gene-specific regulatory mechanism, which silences target gene expression transcriptionally and post-transcriptionally. The RNAi machinery converts the sequence specific information of a long double stranded RNAs (dsRNAs) into small 21-22 nt long dsRNAs (siRNAs, miRNAs) which assemble into an effector complex, the RNA induced silencing complex (RISC). RISC assembly is asymmetric; one strand of an siRNA or a miRNA preferentially incorporates into the RNA-protein complex. Here, I review the rules of the asymmetric RISC formation and discuss their possible regulatory function in several steps in RNAi.

Animals↗
PubMed2005

RNAi-directed assembly of heterochromatin in fission yeast.

Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.

Animals↗
PubMed2006

Assessing RNAi frequency and efficiency in Ophiostoma floccosum and O. piceae.

Ophiostoma species are an economically important group of saprophytic and pathogenic fungi that grow in trees or wood. Ophiostoma like O. piceae and O. floccosum produce melanin, a pigment that stains lumber and logs. We used such species as model organisms for characterizing the molecular mechanisms in fungal melanin production. Because homologous recombination is rare in the Ophiostoma, identifying gene function in this group is challenging. We addressed this by assessing RNA interference (RNAi) as an alternative to gene replacement. For this, we built different inverted repeat transgene (IRT) constructs to down-regulate the polyketide synthase (PKS1) gene of the melanin pathway in O. piceae and O. floccosum. Transformation with IRT-PKS reduced mRNA levels for the PKS1 gene, and consequently decreased pigmentation in transformants. We showed that the PKS1 RNAi efficiency was proportional to the length of the dsRNA expressed from IRT constructs. These results indicated that RNAi is an appropriate tool for functional analysis of genes in Ophiostoma.

Ascomycota↗
PubMed2006

Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs.

RNA interference (RNAi) offers great potential not only for in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene, e.g. in tumor therapy. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradation, a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on (viral) vector delivery may be of only limited clinical use. The more desirable approach is to directly apply catalytically active siRNAs. This review highlights the recent knowledge on the guidelines for the selection of siRNAs which show high activity in the absence of non-specific siRNA effects. It then focuses on approaches to directly use siRNA molecules in vivo and gives a comprehensive overview of in vivo studies based on the direct application of siRNAs to induce RNAi. One promising approach is the in vivo siRNA delivery through complexation of chemically unmodified siRNAs with polyethylenimine (PEI). The anti-tumoral effects of PEI/siRNA-based targeting of tumor-relevant genes in vivo are described.

Gene Silencing↗
PubMed2006

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway.

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.

Antigens, Nuclear↗
PubMed2004

Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis.

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.

Amino Acid Sequence↗
PubMed2005

RNA interference (RNAi) for the silencing of extracellular serine proteases genes in Acanthamoeba: molecular analysis and effect on pathogenecity.

Silencing of extracellular serine protease genes was undertaken by interference RNA (RNAi). Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing the catalytic domain of extracellular serine proteases of Acanthamoeba. In order to confirm the silencing phenomenon, the extracellular serine protease activities in RNAi-treated parasites were compared to non-treated parasites, using zymography profiles, Acanthamoeba-conditioned medium (ACM) protease activity, cytotoxicity assays and extracellular serine protease mRNA levels analysis. Zymography profiles showed a decrease in the extracellular protease levels in the moderate pathogenic and pathogenic strains, after treatment with siRNA. These results were supported after the ACM protease activity and CPE assays were performed in all studied isolates, showing a lower protease activity or cytotoxicity both in the pathogenic and moderate pathogenic strains treated with RNAi. These results support that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba.

Acanthamoeba↗