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BC200 RNA was initially identified as a noncoding RNA of about 200 nt in somatodendritic domains of human nerve cells. It is thought to act as a translational modulator for local protein synthesis. Since BC200 RNA was not observed in somatic cells other than neurons, it was regarded as a neural-specific RNA. However, the neuron-only expression of BC200 does not hold true because it is highly expressed in a number of tumors that are of non-neuronal origin. In this study, we examined biogenesis of BC200 RNA in tumors in detail using cancer cell lines. BC200 RNA was expressed at high levels in several cancer cell lines, but at low levels in normal cell lines. Primer extension analysis and self-ligation RACE revealed that the size of BC200 RNA isolated from cancer cells was heterogeneous, implying the difference of BC200 RNA biogenesis between cancer cells and neural cells. The treatment of actinomycin D, a transcription inhibitor, had little effect on the cellular level of BC200 RNA in cancer cells, but led to the rapid degradation of BC200 RNA in normal cells. This result suggests that BC200 RNA is metabolically unstable in normal cells, while it becomes stable in cancer cells. The increase of stability of BC200 RNA in cancer cells could explain why BC200 RNA was observed at high levels in cancer cells. For cancer cells to maintain high levels of BC200 RNA, therefore, they should have a strategy for RNA protection that normal cells cannot provide. If BC200 RNA plays analogous functional roles in cancer cells as in nerve cells, BC200 RNA may provoke tumorigenesis by repressing translation of tumor suppressing genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4084.

Our understanding of glioblastoma multiforme (GBM), the most common form of primary brain cancer, has been significantly advanced by recent efforts to characterize the cancer genome using unbiased high-throughput sequencing analyses. While these studies have documented hundreds of mutations, gene copy alterations, and chromosomal abnormalities, only a subset of these alterations are likely to impact tumor initiation or maintenance. Furthermore, genes that are not altered at the genomic level may play essential roles in tumor initiation and maintenance. Identification of these genes is critical for therapeutic development and investigative methodologies that afford insight into biological function. This requirement has largely been fulfilled with the emergence of RNA interference (RNAi) and high-throughput screening technology. In this article, the authors discuss the application of genome-wide, high-throughput RNAi-based genetic screening as a powerful tool for the rapid and cost-effective identification of genes essential for cancer proliferation and survival. They describe how these technologies have been used to identify genes that are themselves selectively lethal to cancer cells, or synthetically lethal with other oncogenic mutations. The article is intended to provide a platform for how RNAi libraries might contribute to uncovering glioma cell vulnerabilities and provide information that is highly complementary to the structural characterization of the glioblastoma genome. The authors emphasize that unbiased, systems-level structural and functional genetic approaches are complementary efforts that should facilitate the identification of genes involved in the pathogenesis of GBM and permit the identification of novel drug targets.

RNA interference (RNAi) is an evolutionary conserved mechanism in which a double-stranded RNA (dsRNA) inhibits gene expression by degrading messenger RNA or by blocking the translation pathway of a specific gene.[1] During RNAi, exogenous (e.g., viral RNA) or endogenous (e.g., microRNA) dsRNA is processed and cut to 21- to 23-nucleotide RNA fragments (short interfering RNAs [siRNA]) by a ribonuclease called Dicer and assembled into a RNA-induced silencing complex (RISC). Components of the RISC destroy the passenger strand and recognize and cleave the target mRNA. Since RNAi was first discovered in Canorhabditis elegans a decade ago,[2] it has inspired researchers worldwide to explore more fundamental biological issues and possible biomedical applications The RNAi phenomenon was applied in mammalian cells by two research teams, demonstrating that siRNA can efficiently knock down genes in animal cells.[3, 4] Since then, researchers have been ardently pursuing approaches to manipulating this powerful tool in therapeutics.

Background Mathematical modeling is important to provide insight in the complicated pathway of RNA silencing. RNA silencing is an RNA based mechanism that is widely used by eukaryotes to fight viruses, and to control gene expression. Results We here present the first mathematical model that combines viral growth with RNA silencing. The model involves a plus-strand RNA virus that replicates through a double-strand RNA intermediate. The model of the RNA silencing pathway consists of cleavage of viral RNA into siRNA by Dicer, target cleavage of viral RNA via the RISC complex, and a secondary response. We found that, depending on the strength of the silencing response, different viral growth patterns can occur. Silencing can decrease viral growth, cause oscillations, or clear the virus completely. Our model can explain various observed phenomena, even when they seem contradictory at first: the diverse responses to the removal of RNA dependent RNA polymerase; different viral growth curves; and the great diversity in observed siRNA ratios. Conclusion The model presented here is an important step in the understanding of the natural functioning of RNA silencing in viral infections.

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a method for RNAi in vivo using tissue-specific Gal-4 transgenes to induce dsRNA synthesis from an upstream activator sequence (UAS) vector. This vector contains the desired exonic inverted sequences representing the target gene (preferably more than 400 bp) separated by a unique spacer, the first intron of the actin 5C gene. The inverted repeats are stable during cloning in E. coli with this intronic spacer and the intron is spliced out to produce an almost perfect dsRNA target for Dicer cleavage and the production of siRNAs.

Conventional gene therapy has focused largely on gene replacement in target cells. However, progress from basic research to the clinic has been slow for reasons relating principally to the challenges of heterologous DNA delivery and regulation in vivo. Alternative approaches targeting RNA have the potential to circumvent some of these difficulties, particularly as the active therapeutic molecules are usually short oligonucleotides and the target gene transcript is under endogenous regulation. RNA-based strategies offer a series of novel therapeutic applications, including altered processing of the target pre-mRNA transcript, reprogramming of genetic defects through mRNA repair, and the targeted silencing of allele- or isoform-specific gene transcripts. This review examines the potential of RNA therapeutics, focusing on antisense oligonucleotide modification of pre-mRNA splicing, methods for pre-mRNA trans-splicing, and the isoform- and allele-specific applications of RNA interference.

RNA interference (RNAi) mediated by expression of short hairpin RNAs (shRNAs) is a powerful tool for efficiently suppressing target genes. The approach allows studies of the function of individual genes and may also be applied to human therapy. However, in many instances regulation of RNAi by administration of a small inducer molecule will be required. To date, the development of appropriate regulatory systems has been hampered by the few possibilities for modification within RNA polymerase III promoters capable of driving efficient expression of shRNAs. We have developed an inducible minimal RNA polymerase III promoter that is activated by a novel recombinant transactivator in the presence of doxycycline (Dox). The recombinant transactivator and the engineered promoter together form a system permitting regulation of RNAi by Dox-induced expression of shRNAs. Regulated RNAi was mediated by one single lentiviral vector, blocked the expression of green fluorescent protein (GFP) in a GFP-expressing HEK 293T derived cell line and suppressed endogenous p53 in wild-type HEK 293T, MCF-7 and A549 cells. RNA interference was induced in a dose- and time-dependent manner by administration of Dox, silenced the expression of both target genes by 90% and was in particular reversible after withdrawal of Dox.

Background SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation. Results A PC12 cell line stably transfected with a plasmid expressing an shRNA targeting SNAP-25 has been established. This SNAP-25 knockdown cell line has barely detectable levels of SNAP-25, but normal levels of other synaptic proteins. Catecholamine secretion elicited by depolarization of the SNAP-25 knockdown cells was reduced to 37% of control. Conclusion Knockdown of SNAP-25 in PC12 cells reduces but does not eliminate evoked secretion of catecholamines. Transient expression of human SNAP-25 in the knockdown cells rescues the deficit in catecholamine secretion.

RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression that has demonstrated utility in the inhibition of viral replication. However, the challenges associated with the effective in vivo delivery of siRNAs has been a major obstacle to their use. Here we describe the development of an effective siRNA based therapy for hepatitis virus infection.

Top of pageAbstract The efficient and specific suppression of target genes by RNA interference (RNAi) is a remarkable new approach that may find applications in human therapy whenever genes involved in the respective pathology have to be inhibited. Clinical applications will require a regulatory system that allows control of RNAi by administration of a small inducer molecule. The treatment could then be adapted to the needs of the patient and, should complications arise, the therapy could be stopped or interrupted. RNAi based gene suppression can be induced in target cells by short hairpin RNAs (shRNAs) that are expressed under the control of RNA polymerase III promoters in order to avoid disturbing poly A sequences. To control RNAi by inducible and reversible expression of shRNAs, we developed a novel regulatory system. This system is based on a novel tetracycline-dependent transactivator capable of inducing transcription of shRNAs from a minimal RNA polymerase III promoter in the presence of doxycycline. As a proof-of-principle, tetracycline-controlled RNA interference was used to regulate the expression of GFP in HEK 293T cells stably expressing this transgene. RNA interference was induced by administration of doxycycline yielding a 90% reduction of GFP expression. The effect of doxycycline on the expression of GFP was dose- and time-dependent. In particular, the down-regulation of GFP was reversible after withdrawal of doxycycline, as observed by reappearance of GFP fluorescence.

RNA interference (RNAi) is a robust and highly evolutionarily conserved mechanism for down-regulating gene expression through targeted mRNA cleavage. It has been shown that RNAi can effectively reduce the expression of human immunodeficiency virus 1 (HIV-1) genes in vitro, but since several nucleotides of non-homology between the siRNA and the viral RNA are sufficient to significantly undermine target degradation, the emergence of resistant strains is a major potential obstacle to long term suppression of this highly mutable virus. We seek to identify quantitative design strategies for using RNAi to suppress HIV-1 in a manner that minimizes the chance of viral escape. In the results presented here, we have focused on RNAi strategies that target the viral TAR loop - a very highly conserved non-coding sequence that participates in a positive feedback loop with the viral early transcription factor, Tat.

In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments. Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility. This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.

RNA interference (RNAi) is a natural process, originally described in Caenorhabditis elegans and plants, initiated by the cleavage of long double stranded RNA (dsRNAs) by the ribonuclease III Dicer generating short interfering RNAs (siRNAs). siRNAs induce selective degradation of mRNAs that are homologous to their sequence. In mammalian cells, RNAi interference can be achieved by transfection of chemically synthesized siRNAs or short-hairpin RNA (shRNAs) precursor expressing plasmids. In-vivo gene silencing of endogenous and exogenous genes has been proven by hydrodynamics injection of both shRNAs and synthetic siRNAs, or by using shRNA expressing viral vectors such as lentivirus or adenoviruses.

Top of pageAbstract RNA interference (RNAi) is an innate cellular process in which double-stranded small interfering RNAs (siRNAs) act as triggers to induce sequence-specific degradation of RNA molecules with which they share homology. RNAi has been shown to effectively reduce the expression of viral genes in vitro, which suggests that it might be especially useful as a genetic therapy to combat or protect against viral pathogens that have RNA genomes, such as human immunodeficiency virus (HIV). However, the RNAi response is so specific that several nucleotides of non-homology between the siRNA and the viral RNA are sufficient to significantly impede target degradation. Since HIV mutates rapidly, the emergence of resistant strains is a major obstacle to potential clinical applications. Using an integrated computational and experimental approach, we are studying HIV evolution in response to RNAi pressure in order to develop quantitative design strategies that delay or prevent viral escape. We constructed a novel stochastic simulation that mechanistically models HIV replication, and we used such simulations to make some counterintuitive predictions about the efficacies of several antiviral RNAi strategies. Design considerations we have investigated computationally include the number of HIV sequences targeted, the uniformity of siRNA delivery, and the consequences of targeting the tat-TAR feedback loop. To test these hypotheses experimentally, we developed a lentiviral siRNA vector that induces sustained and dose-dependent reductions (up to >95%) of target gene expression. With this vector, we can effectively and specifically target highly conserved regions of the HIV genome for RNAi-mediated degradation. This system was used to create stable cell lines that can now serve as tunable RNAi challenges for a replicating HIV model virus. We anticipate that these studies will be useful for identifying antiviral RNAi therapy strategies that most effectively suppress HIV infections.

RNA interference or RNAi is a recently characterized mechanism of eukaryotic gene regulation in which a short sequence of double-stranded RNA (dsRNA) specifically down-regulates expression of the associated gene. Preliminary characterization of this phenomenon has revealed a set of inter-related cellular pathways which appear to represent both a response to foreign RNA and a mechanism of endogenous gene regulation. Introduction of dsRNA into cells by a variety of means, including transfection of synthetic RNA duplexes, triggers the RNAi response resulting in specific suppression of target gene expression. Recent efforts on a genome wide scale have involved application of RNAi as an important new tool in cell biology to elucidate gene function in living cells.

RNA interference (RNAi) is the sequence-specific degradation of mRNA by short double-stranded RNA molecules. The technology, introduced only 5 years ago, has stimulated many fantasies regarding the future of functional gene analysis and gene therapy. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all areas of biomedicine. Despite its potential, the major obstacle to the use of RNAi (as for all previous gene silencing approaches) is the need for efficient and sustained delivery of small interfering RNA into primary mammalian cells, and specific targeting of particular cell types in vivo.

AIM: To synthesize three small-interference RNAs (siRNAs) by T7 RNA polymerase-catalyzed reaction, and to investigate their efficacy on modulating the expression of serine/threonine kinase Pim-2 in human colon cancer cell line. METHODS: siRNA I, II and III were synthesized by T7 RNA polymerase-directed in vitro transcription, then transfected into human colon cancer cells SW-480. After incubation for 6 h at 37, 100 mL/L FBS in RPMI 1640 was substituted in each well. After the transfection was repeated twice to three times in each kind of siRNA, hPim-2 mRNA and protein expression were measured by RT-PCR and Western blotting, respectively. RESULTS: Compared to the control group, after transfected for 48 h with hPim-2 siRNA I, II and III, the relative inhibition rates of hPim-2 mRNA expression in colon cancer cells were 65.4% (P < 0.05), 46.2% (P < 0.05) and 56.1% (P < 0.05), respectively. The protein level of hPim-2 was decreased at 72 h compared to the untransfected cells. The relative inhibition percentages of hPim-2 protein by siRNA I, II, III were 61.6% (P < 0.05), 45.8% (P < 0.05) and 55.6% (P < 0.05), respectively. CONCLUSION: The in vitro transcribed siRNAs can be useful for silencing oncogene hPim-2 expression specifically and efficiently. This may open a new path toward the use of siRNAs as a gene-specific therapeutic tool.

Reports that short hairpin RNAs (shRNAs) expressed from plasmids could also trigger RNA interference (RNAi) offered the promise of RNAi gene therapy using viral vectors.1 Two manuscripts by Rubinson et al2 and Hemann et al3 in Nature Genetics deliver on this promise by demonstrating that RNAi delivered by retroviral and lentiviral vectors can silence genes in mice.

Recent discoveries suggest that nature employs a novel approach for the regulation of gene expression by the silencing of endogenous genes. This phenomenon referred to as RNA interference (RNAi) is a posttranscriptional gene-silencing strategy that involves the intermediacy of double-stranded RNA (dsRNA) that complex with and destroy a target messenger RNA (mRNA) (1-4). The targeting and destruction of mRNA has been demonstrated to be highly specific and more selective compared to the antisense approach. Furthermore, it has also been shown that exogenously administered 21- to 23-mer dsRNA, referred to as small interfering RNA (siRNA), is equally efficient as a gene-silencing tool compared to the much longer dsRNA. These discoveries have led to the exciting possibility of using RNAi not only as a potential tool for elucidating the function of genes and analyzing gene expression, but also as a novel therapeutic modality. This review will highlight various aspects of this rapidly advancing field.

RNA interference (RNAi) is a recently discovered, but nevertheless ancient system to block out gene expression. The mechanism of this post-transcriptional gene-silencing effect is based on short interfering RNA molecules (siRNA) of 21–23 base pairs in length, which are generated by the digestion of long double-stranded RNAs by RNase enzymes. The siRNA then guides an endonuclease complex to target mRNAs of homologous sequences resulting in their destruction. This system is presumed to have evolved as a defense mechanism of cells against double-stranded RNA viruses.Novina et al. [1xsiRNA-directed inhibition of HIV-1 infection. Novina, C.D. et al. Nat. Med. 2002; 8: 681–686PubMedSee all References[1] have now evaluated the potential of this system in inhibiting viral infection and replication, particularly of HIV-1. By targetting CD4 – the principal HIVreceptor – with a specific siRNA, an eight-fold decrease in CD4 expression and a consequent four-fold decrease in viral entry was obtained in CD4-expressing non-immune cells (HeLa). In a second approach, delivery of siRNA against the p24 region of the RNA molecule encoding the viral structural Gag proteins into subsequently HIV-1-infected cells resulted in reduced viral protein levels. As the gag gene is part of the viral genomic RNA, the amount of these transcripts is also diminished in p24-siRNA-treated HIV-producing cells. Interestingly, production of virus particles is silenced to a greater extent than the shut down of viral gene expression.In comparable experiments using a human T-cell line, the authors observed similar results. The T cells, however, require a ten-fold higher concentration of siRNA. This might be because of a lower transfection rate of the T cells or a less-efficient silencing mechanism in these cells. The duration of the decreased viral protein synthesis and virus production in Tcells lasts for at least five days after siRNA addition. By day nine, however, the inhibitory effect of the siRNA was no longer detectable.Is siRNA-directed silencing only able to act on viral pre-integration genomes at a very early stage of infection, as shown in the previously mentioned experiments, or also on mRNA from cells with already established infections? Actually, both can happen, as transfection of p24-siRNA into pre-infected cells results in a 50% reduction of p24 expression. The inhibitory effect of p24-siRNA in latently infected T cells stimulated with phorbol ester to produce HIV is even higher.Novina et al. provide in vitro proof of principle of RNAi as a potential tool for the therapy of HIV infections. Still, a major obstacle that must be overcome is the development of efficient systems for the delivery of such siRNAs into relevant target cells in vivo, for instance by the use of viral vectors.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.

Influenza virus defective interfering (DI) RNAs, which originate from polymerase genes by simple internal deletion, can be transcribed in vitro. These DI RNA transcripts contain covalently linked polyadenylic acid, and their synthesis is dependent on ApG or capped RNAs as primers. Furthermore, like the standard viral RNA transcripts, they are complementary in nature and are slightly smaller in size compared with the corresponding DI RNAs. Hybridization of the specific DI RNA transcripts with the corresponding DI RNA segments and analysis of the duplex RNA by gel electrophoresis indicate that they are not incomplete polymerase gene transcripts, but rather the transcripts of the DI RNAs. Since influenza virus DI RNAs contain both the 5' and the 3' termini and transcribe polyadenylic acid-containing complementary RNAs in vitro the mechanism of interference may differ from that of the 5' DI RNAs of Sendai and vesicular stomatitis viruses.

Eukaryotic messenger RNAs and other RNAs containing a fully methylated cap structure (m 7 GpppX m ) stimulate the transcription of influenza viral RNA in vitro . RNA fragments containing the cap and the ensuing 10–14 nucleotides are cleaved from the mRNA and are used as primers to initiate viral RNA transcription. In the infected cell a similar priming by capped host cell RNAs is found.

The study of adenovirus types 2 and 5 viral nuclear RNAs present at early times during a productive infection was analyzed by hybridization of pulse-labeled RNA to restriction fragments of Ad2 and Ad5 DNA. The RNA studied was the total nuclear infected cell population and the double-stranded nuclear RNAs formed after self-annealing, RNase digestion, and selection by Sephadex chromatography. In addition, the viral RNAs were synthesized in the presence and absence of cycloheximide (to restrict the infection to the early phase) at 32° and 41° (to provide control conditions for the H5ts125 mutant defective in the initiation of viral DNA synthesis at the restrictive temperature). The results of these studies established that (1) transcription of the genome is not qualitatively, but is quantitatively, affected by the use of inhibitors of viral DNA synthesis, (2) all regions of the genome are abundantly transcribed including those regions that do not code for early viral mRNA, and (3) most of these non-early messenger RNAs (which include both late mRNA and anti-sense sequences) have been post-transcriptionally modified by polyadenylation. Additionally, liquid hybridization studies of early Ad2 viral nuclear RNA to separated strands of Ad2 Eco (R1-A) fragment DNA indicate that all of these sequences (both sense and anti-sense) are represented in the RNA population and that neither the l nor r strands are preferentially transcribed. All of these results show that self-complementary RNA transcription and the transcription of non-early mRNAs is an integral part of the productive infection of human KB cells by adenovirus 2 and 5. Some of the possible functions of these sequences are discussed.

The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.

Virus specific RNA comprises 5% of the nuclear RNA and 0.5–1.0% of the cytoplasmic RNA of cells transformed by murine sarcoma viruses. Even cryptically transformed cells which possess no detectable virus or viral proteins synthesize detectable amounts of viral RNA.

Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.

Various types of synthetic oligonucleotide duplexes against the Photinus luciferase gene were tested on their induction of the sequence-specific RNA interference (RNAi) activity in transfected human cells. Results indicate that RNA duplexes with ribonucleotide 3′ overhangs rather than those with deoxyribonucleotide 3′ overhangs induce more efficient RNAi activity, and that sense-stranded DNA/antisense-stranded RNA hybrids induce a moderate RNAi activity. These results suggest that there is a difference in the potential of oligonucleotide duplexes to be RNAi mediators, i.e. short interfering RNAs (siRNAs), between human RNAi and invertebrate RNAi. The data further show that different siRNAs induce different levels of RNAi.

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5′ ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

It has been known for some time that plants and insects use RNA interference (RNAi) as nucleic acid-based immunity against viral infections. However, it was unknown whether mammalian cells employ the RNA interference pathway as an antiviral mechanism as well. Over the past years, it has become clear that a variety of viruses, first in plants but recently in insect and mammalian viruses as well, encode suppressors of the RNAi pathway arguing for an antiviral role of this machinery. More recent findings have revealed that certain viruses encode their own microRNAs or microRNA-like RNA molecules, which are processed by the mammalian RNAi machinery. Furthermore, host-encoded microRNAs have been shown to both silence and enhance intracellular levels of viral RNAs. These findings argue that interactions between the RNAi pathway and viral genomes can profoundly affect the outcomes of the viral life cycles and contribute to the pathogenic signatures of the infectious agents.

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO-K1) as well, although double-stranded RNA required for the latter was 2500 times more than for the former. Cognate double-stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.

A new form of circuitry for gene regulation has been identified in which RNAs can crosstalk by competing for shared microRNAs (miRNAs). Such competing endogenous RNAs (ceRNAs) form a network via shared miRNA response elements (MREs) to antagonize miRNA function. We previously reported natural antisense RNA (AS) as an important modulator of interferon-α1 (IFN-α1) mRNA levels by promoting IFN-α1 mRNA stability. We show that IFN-α1 AS forms a ceRNA network with specific IFN-α AS (IFN-α7/-α8/-α10/-α14) and mRNA (IFN-α8/-α10/-α14/-α17) subtypes from the IFN-α gene (IFNA) family to antagonize miRNA-1270 (miR-1270), thereby modulating IFN-α1 mRNA levels. Bioinformatic analysis demonstrated that IFN-α1 AS harbors multiple miR-1270 MREs (MRE-1270s), whose presence was substantiated by miR-1270 overexpression and transfection of antimiR-1270. The antimiR-1270, complementary to the miR-1270 seed region, revealed that IFN-α1 AS likely shares the MRE-1270 with IFN-α1 mRNA and specific IFN-α AS and mRNA subtypes. Subsequent bioinformatic analysis for MRE-1270s showed that IFN-α1 AS and other RNA subtypes shared the 6-mer MRE-1270 site. Further MRE-mapping demonstrated that the total number of MRE-1270s in IFN-α1 AS accounted for approximately 30 % of the miR-1270 population. AntimiR-1270 transfection also caused specific de-repression of five cellular mRNAs, including that of CAPRIN1. These results suggest that IFN-α1 AS, together with specific IFN-α AS and mRNA subtypes, as well as the five cellular mRNAs, participate as competing molecules in the ceRNA network against miR-1270. This coordinated regulatory architecture suggests a vital function for the innate immune system in maintaining precise physiological type I IFN levels via post-transcriptional regulatory mechanisms. Electronic supplementary material The online version of this article (doi:10.1007/s00018-015-1875-5) contains supplementary material, which is available to authorized users.

Two genetically distinct retroviral RNAs can be co-packaged if the RNAs are co-expressed in virion producing cells. For Moloney murine leukemia virus (MLV), co-packaged RNAs are not randomly selected from among all packaging-competent RNAs, but instead primarily associate as homodimers. Here, we tested the hypothesis that the distance between proviral templates might hinder RNA heterodimerization, thus generating the observed preferential homodimerization of co-expressed MLV RNAs. To do this, two genetically distinct RNAs were co-expressed from a single locus and the proportions of hetero- and homodimeric virion RNAs were determined. Unlike RNAs transcribed from two different templates, RNAs transcribed from a single locus dimerized at random. Additionally, in vitro transcription experiments suggested that MLV RNA dimerization can occur more efficiently for longer RNAs during transcription than post-synthesis. Together, these findings show that MLV RNA dimer-partner selection likely occurs either co-transcriptionally or within a pool of transcripts near the proviral template.

Each clone of ts-52 and ts+ WSN influenza virus, when serially passaged at high multiplicity, gives rise to defective interfering (DI) virus with a unique set of new RNA species. The new RNAs (DI RNA) from several DI viruses were examined by the technique of RNase T1 oligonucleotide two-dimensional electrophoresis. It was found that each DI RNA arises from a specific segment of standard viral RNA. All DI RNA studied arose from the viral polymerase genes (P1, P2, and P3). DI RNAs originating from the same polymerase gene were interrelated. Certain of these DI RNAs appeared to contain completely overlapping nucleotide sequences. Others contained both overlapping and nonoverlapping nucleotide sequences. The latter DI RNAs may be formed from the progenitor viral RNA segment by a mechanism other than a common initiation (or termination) point and a simple deletion from one end.