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Microbiology and Molecular Biology... Dec 2003Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a... (Review)
Review
Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.
Topics: Animals; Humans; RNA Interference; RNA, Double-Stranded; RNA, Small Interfering
PubMed: 14665679
DOI: 10.1128/MMBR.67.4.657-685.2003 -
Methods in Molecular Biology (Clifton,... 2018RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a... (Review)
Review
RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a tool for knocking down the expression of individual genes posttranscriptionally, RNAi has been widely used to study the cellular function of genes. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Potential challenges and problems associated with the siRNA technology are also discussed.
Topics: Animals; Electroporation; Gene Knockdown Techniques; Humans; RNA Interference; RNA Stability; Transfection
PubMed: 29423805
DOI: 10.1007/978-1-4939-7471-9_16 -
Cell Death & Disease Jul 2022The growing understanding of RNA functions and their crucial roles in diseases promotes the application of various RNAs to selectively function on hitherto "undruggable"... (Review)
Review
The growing understanding of RNA functions and their crucial roles in diseases promotes the application of various RNAs to selectively function on hitherto "undruggable" proteins, transcripts and genes, thus potentially broadening the therapeutic targets. Several RNA-based medications have been approved for clinical use, while others are still under investigation or preclinical trials. Various techniques have been explored to promote RNA intracellular trafficking and metabolic stability, despite significant challenges in developing RNA-based therapeutics. In this review, the mechanisms of action, challenges, solutions, and clinical application of RNA-based therapeutics have been comprehensively summarized.
Topics: RNA Interference; RNA, Small Interfering
PubMed: 35871216
DOI: 10.1038/s41419-022-05075-2 -
Annual Review of Entomology Jan 2020The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been... (Review)
Review
The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome-assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations.
Topics: Animals; Genetic Techniques; Insecta; RNA Interference
PubMed: 31610134
DOI: 10.1146/annurev-ento-011019-025224 -
The EMBO Journal Nov 2023RNA-based therapeutics have the potential to revolutionize the treatment and prevention of human diseases. While early research faced setbacks, it established the basis... (Review)
Review
RNA-based therapeutics have the potential to revolutionize the treatment and prevention of human diseases. While early research faced setbacks, it established the basis for breakthroughs in RNA-based drug design that culminated in the extraordinarily fast development of mRNA vaccines to combat the COVID-19 pandemic. We have now reached a pivotal moment where RNA medicines are poised to make a broad impact in the clinic. In this review, we present an overview of different RNA-based strategies to generate novel therapeutics, including antisense and RNAi-based mechanisms, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Using three rare genetic diseases as examples, we highlight the opportunities, but also the challenges to wide-ranging applications of this class of drugs.
Topics: Humans; RNA; Pandemics; Gene Editing; RNA Interference; Genetic Therapy
PubMed: 37728251
DOI: 10.15252/embj.2023114760 -
RNA (New York, N.Y.) Apr 2003Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila...
Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.
Topics: Dendritic Cells; Genes, Reporter; HeLa Cells; Humans; In Vitro Techniques; Lentivirus; RNA Interference
PubMed: 12649500
DOI: 10.1261/rna.2192803 -
Viruses Feb 2022Multiple antiviral immunities were developed to defend against viral infection in hosts. RNA interference (RNAi)-based antiviral innate immunity is evolutionarily... (Review)
Review
Multiple antiviral immunities were developed to defend against viral infection in hosts. RNA interference (RNAi)-based antiviral innate immunity is evolutionarily conserved in eukaryotes and plays a vital role against all types of viruses. During the arms race between the host and virus, many viruses evolve viral suppressors of RNA silencing (VSRs) to inhibit antiviral innate immunity. Here, we reviewed the mechanism at different stages in RNAi-based antiviral innate immunity in plants and the counteractions of various VSRs, mainly upon infection of RNA viruses in model plant Arabidopsis. Some critical challenges in the field were also proposed, and we think that further elucidating conserved antiviral innate immunity may convey a broad spectrum of antiviral strategies to prevent viral diseases in the future.
Topics: Arabidopsis; Host-Pathogen Interactions; Immunity, Innate; Plant Diseases; Plant Immunity; RNA Interference; RNA Virus Infections
PubMed: 35216025
DOI: 10.3390/v14020432 -
Current Opinion in Immunology Oct 2018Infection of plants and insects with RNA and DNA viruses triggers Dicer-dependent production of virus-derived small interfering RNAs (vsiRNAs), which subsequently guide... (Review)
Review
Infection of plants and insects with RNA and DNA viruses triggers Dicer-dependent production of virus-derived small interfering RNAs (vsiRNAs), which subsequently guide specific virus clearance by RNA interference (RNAi). Consistent with a major antiviral function of RNAi, productive virus infection in these eukaryotic hosts depends on the expression of virus-encoded suppressors of RNAi (VSRs). The eukaryotic RNAi pathway is highly conserved, particularly between insects and mammals. This review will discuss key recent findings that indicate a natural antiviral function of the RNAi pathway in mammalian cells. We will summarize the properties of the characterized mammalian vsiRNAs and VSRs and highlight important questions remaining to be addressed on the function and mechanism of mammalian antiviral RNAi.
Topics: Animals; Humans; Mammals; Protective Agents; RNA Interference; RNA, Viral; Virus Diseases; Viruses
PubMed: 30015086
DOI: 10.1016/j.coi.2018.06.010 -
Nature Cell Biology Dec 2022The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the...
The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.
Topics: RNA Interference; Endoplasmic Reticulum
PubMed: 36471127
DOI: 10.1038/s41556-022-01025-4 -
Microbiology Spectrum Dec 2016RNA interference (RNAi) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding RNAs (sRNAs) of about 20 to... (Review)
Review
RNA interference (RNAi) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding RNAs (sRNAs) of about 20 to 30 nucleotides. Its discovery is one of the most important scientific breakthroughs of the past 20 years, and it has revolutionized our perception of the functioning of the cell. Initially described and characterized in Neurospora crassa, the RNAi is widespread in fungi, suggesting that it plays important functions in the fungal kingdom. Several RNAi-related mechanisms for maintenance of genome integrity, particularly protection against exogenous nucleic acids such as mobile elements, have been described in several fungi, suggesting that this is the main function of RNAi in the fungal kingdom. However, an increasing number of fungal sRNAs with regulatory functions generated by specific RNAi pathways have been identified. Several mechanistic aspects of the biogenesis of these sRNAs are known, but their function in fungal development and physiology is scarce, except for remarkable examples such as Mucor circinelloides, in which specific sRNAs clearly regulate responses to environmental and endogenous signals. Despite the retention of RNAi in most species, some fungal groups and species lack an active RNAi mechanism, suggesting that its loss may provide some selective advantage. This article summarizes the current understanding of RNAi functions in the fungal kingdom.
Topics: Fungi; Gene Expression Regulation, Fungal; RNA Interference; RNA, Fungal
PubMed: 28087943
DOI: 10.1128/microbiolspec.FUNK-0008-2016