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RNA plays important and diverse roles in biology, but molecular tools to manipulate and measure RNA are limited. For example, RNA interference (RNAi)1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here, we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8 (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of fifteen orthologs in E. coli, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts. We demonstrate that LwaCas13a is capable of providing comparable levels of knockdown as RNAi, but with dramatically improved specificity. Moreover, catalytically inactive LwaCas13a maintains targeted RNA binding, allowing for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for RNA targeting with wide applicability for studying RNA in mammalian cells.

Review describes the phenomenon of RNA interference – the recently discovered biological mechanism of the negative regulation of gene expression. The mechanism is based on the block of the translation and/or degradation of messenger RNA under short non-coding RNA, among them: microRNA and small interfering RNA. The paper reviews the molecular processes of the formation of small RNA, the mechanism of their action and the feasibility of small RNA implementation in the anti-tumor therapy. Specially, we analyze the approaches to in vivo delivery of small RNA, in particular – the liposome and exosome constructs, and perspectives of the involvement of such constructs in the cancer treatment.

mRNA is an attractive drug target for therapeutic interventions. In this review we highlight the current state, clinical trials, and developments in antisense therapy, including the classical approaches like RNaseH-dependent oligomers, splice-switching oligomers, aptamers, and therapeutic RNA interference. Furthermore, we provide an overview on emerging concepts for using RNA in therapeutic settings including protein replacement by in-vitro-transcribed mRNAs, mRNA as vaccines and anti-allergic drugs. Finally, we give a brief outlook on early-stage RNA repair approaches that apply endogenous or engineered proteins in combination with short RNAs or chemically stabilized oligomers for the re-programming of point mutations, RNA modifications, and frame shift mutations directly on the endogenous mRNA.

The ability to develop efficient and versatile technologies for manipulating gene expression is a fundamental issue both in biotechnology and therapeutics. The endogenous RNA interference (RNAi) pathway which mediates gene silencing was discovered at the end of the 20th century and it is nowadays considered as an essential strategy for knockdown of specific genes and for studying gene function. Remarkably, during the past decade, a RNA-induced mechanism of gene activation has also been reported. Likewise RNAi, the RNA activation (RNAa) process is also mediated by sequence-specific double-stranded RNA (dsRNA) molecules, and interesting resemblances between both RNA-based transcriptional mechanisms have been described. Small activating RNAs (saRNAs) and related molecules have been used for targeting of genes in species that are as different as nematodes and humans, and similar dsRNA-induced activation phenomena have also been observed in plants. The aim of this letter is to highlight recent molecular insights into yet unexplored RNAa mechanism and its potential for manipulating transcriptional activity. Fil: Vaschetto, Luis Maria Benjamin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina

The review intends to present and recapitulate the current knowledge on the roles and importance of regulatory RNAs, such as microRNAs and small interfering RNAs, RNA binding proteins and enzymes processing RNAs or activated by RNAs, in cells infected by RNA viruses. The review focuses on how non-coding RNAs are involved in RNA virus replication, pathogenesis and host response, especially in retroviruses HIV, with examples of the mechanisms of action, transcriptional regulation, and promotion of increased stability of their targets or their degradation.

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3’UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells., eLife digest Cells store their genetic code within molecules of DNA. Some of this information will be copied into chemically similar molecules called RNAs, from which the sequence of letters in the genetic code can be translated to build proteins. However, these messenger RNAs are not the only RNA molecules that cells can make. MicroRNAs are other short pieces of RNA that closely match sequences in parts of certain messenger RNAs. The messenger RNAs targeted by microRNAs are broken down inside the cell, which reduces how much protein can be produced from them. Since its discovery, scientists have exploited this process – called RNA interference (or RNAi for short) – and designed microRNA-like small interfering RNAs (siRNAs) to target particular messenger RNAs and decrease the levels of the corresponding proteins in countless experiments. Two proteins that have been studied in RNAi experiments are CD95 and its interaction partner CD95L. Both of these proteins are important in human cancer cells, and targeting them via RNAi killed cancer cells in an unknown mechanism that the cancer cells were unable to resist. RNAi experiments are designed to be specific, but sometimes they can accidently target other non-target messenger RNAs. Putzbach, Gao, Patel et al. have now analyzed all of the siRNAs that can be made from the messenger RNAs for CD95 and CD95L to mediate RNAi in cancer cells. This revealed that several messenger RNAs, other than those for CD95 and CD95L, were unintentionally being targeted, including many that code for proteins that cells need to survive. Further examination of the messenger RNA for CD95 and CD95L showed that they contain short sequences that are similar to those in the messenger RNAs of the genes that encode these survival proteins. Putzbach et al. were able to study and then predict which siRNA sequences would be toxic to cancer cells. These findings indicate that an RNAi off-target effect may actually be used to kill cancer cells. Future studies will determine whether this effect could be exploited to shrink tumors in animal models of cancer. If successful, this in turn could lead to new treatments for cancer patients.

RNA interference (RNAi) is a naturally occurring mechanism for gene silencing induced by the presence of short interfering RNA (siRNA). RNAi is an endogenous catalytic pathway that is triggered by double-stranded RNA (dsRNA). The trigger can occur naturally, as in the case of a cellular infection by a dsRNA virus, or by the intentional introduction of dsRNA to induce user-directed degradation of the cognate transcript(s). RNAi is fundamentally a two-step process. The first step involves the master enzyme in the RNAi process, a type III endoribonuclease aptly named Dicer. This enzyme is involved in the ATP-dependent digestion of longer dsRNA into 21–23 bp siRNA molecules with characteristic 3′ dinucleotide overhangs on both strands. In the second step, siRNA, regardless of the source, becomes part of a multicomponent RNA-induced silencing complex (RISC). The use of RNAi as a means of studying the effects of gene expression in a cell or in an organism is occasionally called reverse genetics, the goal of which is to determine the consequences for a cell when a protein is not produced. In other words, the function of a gene can often be discovered by silencing it. The overall RNAi efficiency in an experiment is also a function of the abundance and the stability of both the target mRNA and the encoded protein, and the precise region of the mRNA targeted for scission. CRISPR (clustered regular interspersed short palindromic repeats) is a natural defense mechanism that evolved in prokaryotes to protect themselves against viral DNA. From a contemporary molecular biology perspective, there are two useful things that the CRISPR–Cas9 system can be programmed to do in this regard: silence a gene or edit a gene. In the case of the former, the guide RNA leads the CRISPR–Cas9 complex to the target gene and then mediates cutting the gene on both strands. Although cells attempt to repair the gene, including re-ligation, the process is error-prone and introduces mutations. The net result is that the gene becomes nonfunctional and is effectively silenced. In contrast, when a repair template is made available during the attempted gene repair process, the new information that the repair template carries becomes incorporated into the DNA such that the target gene is either repaired (if it was defective) or acquires a new function.

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens1. However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago2, remains unknown3. Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence4-7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)8,9. Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice10,11. However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells12-21. Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Silencing of gene expression by RNA interference(RNAi)has become a widely used tool for assessing gene function in a fast and easy manner. An important advance in the RNAield was the discovery that plasmid-based short hairpin RNA transcription can substitute for synthetic small interference RNAs both in vitro and in vivo. The constitutive knockdown of gene expression by RNAi can limit the scope of experiments, especially if the inhibition of genes leads to cell lethality, which prevents in vivo functional analysis. We have generated double-conditional short hairpin RNA from a single vector that can regulate its own transcription by a combination of tetracycline-inducible and Cre-loxP systems. This vector will be useful for the control of short hairpin RNA expression in a spatially-regulated manner by Cre recombinase as well as a temporally-regulated manner by tetracycline induction. These control features provide for the specic silencing of genes to facilitate functional genetic analysis in mammals in vivo.

Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

RNAi using single-strand RNA would provide new options for therapeutic development and for investigating critical questions of mechanism. Using chemically modified single-strands, we test the hypothesis that single-stranded RNAs can engage the RNAi pathway and silence gene transcription. We find that a chemically modified single-stranded silencing RNA (ss-siRNA) designed to be complementary to a long noncoding RNA (lncRNA) requires argonaute protein, functions through the RNAi pathway, and inhibits gene transcription. These data expand the use of single-stranded RNA to cell nuclei.

Small RNAs are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. They are almost exclusively used to silence gene expression post-transcriptionally through degradation of mRNA. Small RNAs, however, can have a broader range of function by binding to Argonaute proteins and associating with complementary RNA targets in the nucleus, including long noncoding RNAs (lncRNAs) and pre-mRNA. Argonaute-RNA complexes can regulate nuclear events like transcription, genome maintenance, and splicing. Thousands of lncRNAs and alternatively spliced pre-mRNA isoforms exist in humans, and these RNAs may serve as natural targets for regulation and therapeutic intervention. This review describes nuclear mechanisms for Argonaute proteins and small RNAs, new pathways for sequence-specific targeting, and the potential for therapeutic development of small RNAs with nuclear targets.

Gene silencing by RNA triggers is an ancient, evolutionarily conserved, and widespread phenomenon. This process, known as RNA interference (RNAi), occurs when double-stranded RNA helices induce cleavage of their complementary mRNAs. Because these RNA molecules can be introduced exogenously as small interfering RNAs (siRNAs), RNAi has become an everyday experimental tool in laboratory research. In addition, the number of RNA-based therapeutics that are currently in clinical trials for a variety of human diseases demonstrate the therapeutic potential of RNAi. In this Account, we focus on our current understanding of the structure and function of various classes of RNAi triggers and how this knowledge has contributed to our understanding of the biogenesis and catalytic functions of siRNA and microRNA in mammalian cells. Mechanistic studies to understand the structure and function of small RNAs that induce RNAi have illuminated broad functions of the ancient RNAi machinery in animals and plants. In addition, such studies have provided insight to identify endogenous physiological gene silencing RNA triggers that engage functional machineries similar to siRNAs. Several endogenous small RNA species have been identified: small noncoding RNAs (microRNAs), piwi-interacting RNAs (piRNAs), and endogenous siRNAs (endo-siRNAs). microRNAs are the most widespread class of small RNAs in mammalian cells. Despite their importance in biology and medicine, the molecular and cellular mechanisms of microRNA biogenesis and function are not fully understood. We provide an overview of the current understanding of how these molecules are synthesized within cells and how they act on gene targets. Interesting questions remain both for understanding the effects of modifications and editing on microRNAs and the interactions between microRNAs and other cellular RNAs such as long noncoding RNAs.

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon β. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19–27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25–28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8–10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.

MicroRNAs (miRNA) have emerged as critical player in the field of life sciences. These are endogenously expressed small non coding RNAs that function to control post-transcriptional gene expression. These small RNAs have been shown to regulate the cell growth, differentiation and apoptosis. miRNAs regulate messenger RNA expression by targeting homologous sequences in mRNAs and further regulate the expression of endogenous genes either by mRNA cleavage or translational repression. One miRNA is capable of regulating several distinct mRNAs and are believed to modulate more than one-third of the mRNA species encoded in the human genome. In this review, biogenesis of miRNAs and their regulatory mechanism is addressed. Moreover, the potential application of miRNA in the field of development and disease is highlighted. Computational and experimental approaches to identify novel miRNA, its expression and targets are also explained. The complex nature of miRNAs remains a mystery and their role in RNA interference makes it a potential target for future studies.

Delivery of RNA-based therapeutics, for example RNA interference (RNAi) effectors, to target cells is one of the major obstacles for the development of RNA-based therapies. Since it has been known for a long time that bacteria can mediate tumor shrinkage, it was obvious to use nonpathogenic bacteria to produce and deliver therapeutic RNA molecules into target cells to induce RNAi. During the last years, two bacteria-based concepts were developed for this strategy, transkingdom RNAi (tkRNAi) and bacteria-mediated RNAi (bmRNAi). The first concept, tkRNAi, delivers RNAi effectors into target cells by invasive bacteria, which themselves produce therapeutic shRNAs. The bmRNAi technology utilizes invasive bacteria conveying RNAi effector-encoding DNA constructs that will act as a matrix for transcription of these sequences in the target cell by the host cell’s transcription machinery.

Although most bacterial small RNAs act to repress target mRNAs, some also activate messengers. The predominant mode of activation has been seen in 'anti-antisense' regulation whereby a small RNA prevents the formation of an inhibitory 5' mRNA structure that otherwise impairs translational initiation and protein synthesis. The translational activation might also stabilize the target yet this was considered a secondary effect in the examples known thus far. Two recent papers in Molecular Microbiology investigate post-transcriptional activation of collagenase mRNA by Clostridium VR-RNA, and streptokinase mRNA by Streptococcus FasX RNA, to suggest that small RNAs exert positive regulation of virulence genes primarily at the level of mRNA stabilization.

RNA interference, a recently discovered new mechanism controlling gene expression via small RNAs, was shown to be involved in characterization and control of basic ovarian cell functions. The main classes of small RNAs, as well as their expression in ovaries have been described. Furthermore, the successful application of RNA interference for study and control of basic ovarian functions (proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation, and related ovarian cell malignant transformation) and production of recombinant proteins have been demonstrated. Application of RNA interference in reproductive biology and medicine can be successful in two main areas: (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for regulation of reproductive processes and treatment of reproductive disorders or their particular indexes. Problems of improvement of small RNA delivery to target ovarian cells and potent RNA interference-related approaches for treatment of ovarian disorders (especially of ovarian cancer) have been discussed.

RNA transfection methods have not proven to be as popular as DNA methods due to the highly transient nature of the RNA inside the cell. However, there are many advantages in using RNA for gene over-expression, such as the rapidity of expression, the ability to express in all cell types without the need for cell-type specific promoters, and the ability to analyze the effects of gene over-expression in a transient manner. Therefore, we have developed a method (StabiLizingUtr: SLU) to stabilize the RNA for varying durations, using specific sequences from the 3′UTR of the Venezuelan equine encephalitis virus (VEEV). We have designed a plasmid for cloning genes upstream from repeated stabilizing sequences to generate mRNA with one or more VEEV stabilizing sequence motifs. We demonstrate this method in several cell and tissue types, including the mammalian cochlea, a tissue that has been difficult to transfect with other methods.

Cyclin-dependent kinase 2 (CDK2) is a member of serine/ threonine protein kinases, which initiates the principal tran- sitions of the eukaryotic cell cycle and is a promising target for cancer therapy. The present study was designed to inhibit cdk2 gene expression to induce cell cycle arrest and cell pro- liferation suppression. Here, we constructed a series of RNA interference (RNAi) plasmids which can successfully express small interference RNA (siRNA) in the transfected human cells. The results showed that the RNAi plasmids containing the cod- ing sequences for siRNAs down-regulated the cdk2 gene ex- pression in human cancer cells at the mRNA and the protein levels. Furthermore, we found that the cell cycle was arrested at G0G1 phases and the cell proliferation was inhibited by dif- ferent siRNAs. These results demonstrate that suppression of CDK2 activity by RNAi may be an effective strategy for gene therapy in human cancers. (BMB reports 2010; 43(4): 291-296)

Virtually all animals and plants utilize small RNA molecules to control protein expression during different developmental stages and in response to viral infection. Structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small RNA biogenesis, formation of RNA-induced silencing complexes (RISCs), and targeting of complementary mRNAs. Here we review exciting recent progress in understanding how regulatory RNAs are produced and how they trigger specific destruction of mRNAs during RNA interference (RNAi).

Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, which are processed via the RNAi pathway. The chemical modulation of RNAi has important therapeutic relevance, because a wide range of miRNAs has been linked to a variety of human diseases, especially cancer. Thus, the activation of tumor-suppressive miRNAs and the inhibition of oncogenic miRNAs by small molecules have the potential to provide a fundamentally new approach for the development of cancer therapeutics.

RNA interference has been recognized to be an efficient mechanism for the sequence specific inhibition of gene-expression caused by 21–23 bp long dsRNA. This is a potent tool to study gene function and is ready to lend a hand in the treatment of disease. Two main classes of dsRNA involved are short-interfering RNA and micro RNA which differ in biogenesis and modes of target regulation. The discovery of RNAi has led to the development in the field of biomedical research. The evolution of RNAi has led to diverse applications such as identification of gene function, identification of disease causing genes and therapeutics. This homology dependent gene silencing, hence has a great latent in transcriptional regulation and medicine. This review summarizes the role of RNAi in human health and diseases.

In RNAi (RNA interference), siRNAs (small interfering RNAs) are loaded into the RISC (RNA-induced silencing complex), which then mediates endonucleolytic cleavage of complementary target RNAs. Although RNAi has become one of the most powerful tools in molecular biology to assess gene function, there remains a great number of ineffective siRNAs. It is already known that the assembly and activation of RISC is a crucial determinant of RNAi activity, but downstream effects such as target accessibility have not been analysed extensively. Therefore we assessed the effect of target site accessibility and found that it significantly improves the potency of siRNAs. Similarly, miRNAs (microRNAs) act by repressing protein synthesis through imperfect base-pairing to the 3′-UTR (untranslated region) of target mRNAs. We found that predicted target sites reside in regions of high accessibility and tested whether this criterion could be used in the search of functional miRNA targets. In addition, we performed reporter gene assays to test whether accessibility correlates with measured mRNA suppression levels. The results of our initial study suggest that secondary structures might add a so far underrepresented layer of complexity in the recognition of RNA targets by miRNAs.

The recent discovery that small regulatory RNAs have diverse roles in eukaryotic intracellular life, such as fine tuners of endogenous gene expression, as inhibitors of viral replication and as suppressors of transposon activity, is now widely recognized as a major breakthrough in the understanding of cellular biology. The pathways used by these small RNAs seem to build upon a few central mechanisms in which small double-stranded RNAs (dsRNAs) are generated from various precursor RNAs of exogenous or endogenous origin and used for inhibiting expression of genes with sequence complementarity to the small RNA. In particular, one class of small gene-regulatory RNAs, known as small interfering RNAs (siRNAs), has the potential for highly specific gene silencing. These are c. 21 nucleotide perfectly complementary dsRNAs with 3′ end overhangs. They are produced by cleavage of long dsRNA believed to be of viral origin. Subsequently, a large cytoplasmic protein complex known as the RNA-induced silencing complex (RISC) uses the antisense strand of the siRNAs for targeting complementary mRNA strands followed by cleavage of these transcripts in the mechanism called RNA interference (RNAi). Accordingly, synthetic siRNAs may have a potential for treating diseases caused by intracellular pathogens like viruses by specifically targeting the expression of their molecular components. Furthermore, as target sequences need not be of viral origin, siRNAs hold a promise for the treatment of various cellular diseases and as a tool in reverse genetic studies. SiRNAs are currently exploited for such purposes in diverse organisms ranging from plants and invertebrates to human beings. But due to initial difficulties, when using RNAi for gene silencing in fishes, only a few such studies have been conducted in fishes. The purpose of this review is to inspire further experimental work.

Several classes of small RNAs are known in plants and accumulating evidence indicates that different classes of small RNAs may function either cell autonomously or noncell autonomously to regulate gene expression. Here, a simple visual assay used to compare the biological activity of small RNAs

Firstly, we have to thank the Nobel Prize winners of Physiology or Medicine 2006, Drs. Andrew Fire at Stanford University and Craig Mello at the University of Massachusetts Medical School, for their pioneering work in RNA interference (RNAi). Without their discovery, we may not have been able to understand the role of RNAs in regulating gene expression today. RNAi is a process that is initiated by the introduction of RNA into a cell and ends with the degradation of complementary messenger RNA, thus stopping or otherwise affecting gene expression. In this review, we will describe the mechanisms involved in the processing of the RNA and how gene expression can be regulated. Since its discovery, RNAi has been widely studied and therapeutic investigations are ongoing. It is becoming increasingly apparent that RNAi has potential for cancer treatment in the future. This review focuses on the evolving development of the RNAi technique in plants, worms, fruit flies, and mammalian cells and particular attention is...

RNA interference (RNAi) has become one of the most powerful and popular approach on gene silencing in clinical research study especially in virology due to the gene-specific suppression property of small interfering RNA (siRNA). In this report, we demonstrate that expression of vector-mediated small hairpin RNA (shRNA) against human immunodeficiency virus type 1 (HIV-1) integrase (IN), one of the three important enzymes in HIV infection by controlling the integration of viral RNA to host DNA, could suppress the protein synthesis of EGFP-tagged IN in HeLa cell model efficiently. Furthermore, we show that IN shRNA can successfully reduce the HIV particles production in 293T cells at the level similar to the positive control of HIV-1 tat shRNA. These results provide the therapeutic possibility of HIV replication using RNAi against HIV-1 integrase.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation process conserved in fungi, plants and animals. The trigger of the mechanism is double-stranded RNA derived from transgenic or endogenous loci and formed by intra- or inter-molecular interactions of single-stranded RNAs or the action of RNA-dependent RNA polymerases (RDRs). Double-stranded RNA from various sources is processed by one of the four Dicer-like (DCL) proteins in Arabidopsis, and the resulting short RNAs enter into at least four different pathways, one of which involves the production of trans-acting short interfering RNAs (tasiRNAs). We report here a novel gene (SDE5) that is required for transgene silencing and the production of tasiRNAs. Mutation in SDE5 also results in hyper-susceptibility to cucumber mosaic virus but not turnip mosaic virus. However, like RDR6, SDE5 is not involved in inverted repeat-induced transgene silencing or the biogenesis of microRNAs and 24 nt siRNAs produced by DCL3. Based on these results, we propose that SDE5 acts together with RDR6 in generating double-stranded RNA from specific single-stranded RNAs. As the sequence of SDE5 has sequence features shared by TAP, a human mRNA export factor, we propose that its role could be in the transport of RNA molecules that are converted into a double-stranded form by RDR6.

Since some years, activation of RNA interference (RNAi) has been developed into a widely used and powerful biological tool to functionally annotate genomes. Several variants of small regulatory RNAs can trigger this evolutionary highly conserved cellular pathway leading to specific hybridisation to and subsequent degradation or translational repression of target mRNAs. These regulatory RNAs include synthetic double-stranded small interfering RNAs (siRNAs), pol III transcribed small hairpin RNAs (shRNAs), or endogenous or artificial micro RNAs (miRNAs) which are expressed from pol II promoters as primary pri-miRNA transcripts subsequently processed into mature miRNAs in a regulated multi-step process. Depending on the mode of activation RNA-processing and efficacy as well as kinetics of RNAi may differ from transient effects to long-lasting gene silencing. However, one of the major challenges for in vitro and in vivo application of RNAi still remains the efficient delivery of suitable RNAi-triggers to target cells. This review highlights the mechanism of RNAi and its activation, the use of plasmid-based and retro-/ lentiviral vector-based systems to mediate long-term RNAi, kinetic aspects of RNAi and their impact on selection of cell populations with modified gene expression.

RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by small double-stranded RNA homologous in sequence to the target gene. Various factors involved in the regulation of hepatocyte function have been identified using RNAi, indicating that RNAi is a useful strategy for characterization. There has been some success in treating experimental liver dysfunction using RNAi in several model systems, suggesting a promising new therapeutic strategy. A number of groups have also demonstrated that RNAi can interfere with hepatitis C virus and hepatitis B virus gene expression and replication in several model systems, suggesting a new approach for the treatment of these viral diseases. This review summarizes studies of hepatocytes using RNAi.

Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3′ end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3′ overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications., Short hairpin RNAs are widely used to produce small interfering RNAs (siRNAs) for gene silencing. Here, the authors show that an alternative siRNA precursor in the presence of a self-cleaving ribozyme has enhanced silencing activity and reduced off-target effects, providing a potential RNAi tool.

The study of the interaction between RNA and DNA sequences in activating genes has important significance for understanding the mechanisms of RNA-mediated activation. Here, we used in vitro chromatin reconstitution approach to observe whether RNAs increase DNase I digestion, plasmid transfection to observe whether RNAs promote gene expression, and bioinformatics analysis to predict the binding ability of RNAs to centromere DNA (constitutive heterochromatin). Synthetic RNAs (23nt) that were complementary to mouse albumin gene and total liver RNA increased DNase I digestion sensitivity of mouse albumin gene, suggesting that RNAs can increase chromatin accessibility. Transcribed sense-antisense tandem Alu elements activated an enhanced green fluorescent protein reporter gene after stable transfection. Bioinformatics analysis showed that the binding strength of RNA population to centromere DNAs is significantly lower than that of their flanking sequences, which suggests that the centromere is not easily affected by RNAs produced from other transcribed regions and may be the reason why centromeres consist of constitutive heterochromatin. The results in this paper illustrate that RNAs complementary to DNA sequences play roles in activating genes. Since RNA is mainly produced from the cell's own DNA, the work presented in this paper suggests that RNAs transcribed from DNA create feedback that activates DNA transcription.

Objective Short interfering RNA is an effective method for target gene knockdown. However, concerns surround the design, administration, efficacy, specificity, and immunostimulatory potential. Although uptake by alveolar macrophages has been demonstrated, studies have not examined its use in lung ischemia–reperfusion injury. We describe the validation of short interference RNA as a novel technique for cell-specific target gene knockdown in our model of lung ischemia–reperfusion injury. Methods Dose-response experiments were performed, and 3 distinct sequences of toll-like receptor-4, toll-like receptor-2, and myeloid differentiation factor-88 short interference RNA were tested for efficacy of knockdown. Saline, lipid vector, and noncoding short interference RNA controls were used. Similar experiments were performed in primary cultures of resident pulmonary cells. Target protein knockdown was assessed by Western blot. Rat serum and cell culture media were assessed for interferon and cytokine production. Biotin labeling was used to assess short interference RNA uptake. Results Target protein expression was significantly reduced using short interference RNA. However, toll-like receptor-4 knockdown was isolated to alveolar macrophages, and biotin labeling confirmed toll-like receptor-4 short interference RNA localization to alveolar macrophages. There was significant knockdown of toll-like receptor-4 expression in cultured cells treated with toll-like receptor-4 short interference RNA. There was no significant change in interferon production after short interference RNA treatment. There was effective target protein knockdown with each sequence used. Conclusions Short interference RNA is a valid method for achieving target protein knockdown in alveolar macrophages and is an important tool in the evaluation of its role in the development of lung ischemia–reperfusion injury.

RNA interference (RNAi) is an RNA degradation process that involves short, double-stranded RNAs (dsRNA) as sequence specificity factors. The natural function of the RNAi machinery is to generate endogenous short double-stranded RNAs to regulate gene expression. It has been shown that treatment of Plasmodium falciparum, the etiologic agent of malaria, with dsRNA induces degradation of the corresponding microRNA (miRNA), yet typical RNAi-associated genes have not been identifiable in the parasite genome. To clarify this discrepancy we set out to clone short RNAs from P. falciparum-infected red blood cells and from purified parasites. We did not find any short RNA that was not a rRNA or tRNA fragment. Indeed, only known human miRNAs were isolated in parasite preparations indicating that very few if any short RNAs exist in P. falciparum. This suggests a different mechanism than classical RNAi in observations of dsRNA-mediated degradation. Of the human miRNAs identified, the human miRNA mir-451 accumulates at a very high level in both infected and healthy red blood cells. Interestingly, mir-451 was not detectable in a series of immortalised cell lines representing progenitor stages of all major blood lineages, suggesting that mir-451 may play a role in the differentiation of erythroid cells.

Short RNAs (21–27 nt) silence genes that contain homologous nucleotide sequences; this is known as RNA silencing. This review considers the generation of short RNAs from their precursors: double-stranded RNAs, capable of inducing RNA interference, and hairpin RNAs, whose processing yields microRNAs, as well as the properties of RNA-binding domains that were initially identified in proteins operating in RNA interference. The interactions between these domains and known RNA-binding modules within proteins involved in RNA interference and microRNA generation are described.

RNA interference is a mechanism of posttranslational (at the level of mRNA) gene silencing. Sequence-specific mRNA degradation is realized with the help of small interfering RNAs produced by processing of a precursor using Dicer, an enzyme from the RNAse III family. This mechanism is now widely used in vitro on cultures of mammalian cells in order to elucidate functions of individual genes by gene specific knockdown. Analogs of small interference RNAs are intensely expressed during embryogenesis. The mechanism of RNA interference plays an especially important role in embryogenesis of invertebrates. Identification of the functions of small noncoding RNAs is essential for understanding the genetic mechanisms underlying individual developmental stages. In order to integrate small interference RNAs in mammalian cells, various systems have been developed that allow both transient (for 48 h) and stable expression in vitro. These systems are considered in the present review.

RNA interference (RNAi) induces gene silencing at a level of posttranscription mediated by double stranded RNA. There are numerous methods for delivery of small double-stranded interference RNA (siRNA) to the target cells, including nonviral and viral vectors. Among these methods, viral vectors are the more efficient vehicles. The expression of short hairpin RNA (shRNA) by viral vectors in target cells can be cut by Dicer enzyme to become ∼21 bp siRNA, which could guide degradation of cognate mRNA. RNAi technology can be directed against cancer using a variety of strategies, including the inhibition of overexpressed oncogenes, promoting apoptosis, regulating cell cycle, antiangiogenesis and enhancing the efficacy of chemotherapy and radiotherapy. Since RNAi technology has become an excellent strategy for cancer gene therapy, this review outlines the latest developments and applications of such a novel technology.

Silencing of gene expression by ribonucleic acid (RNA), known as RNA interference (RNAi), is now recognized as a major means of gene regulation in biology. In this mechanism, small noncoding double-stranded RNA molecules knock down gene expression through a variety of mechanisms that include messenger RNA (mRNA) degradation, inhibition of mRNA translation, or chromatin remodeling. The posttranscriptional mechanism of RNAi has been embraced by researchers as a powerful tool for generating deficient phenotypes without mutating the gene. In parallel, exciting recent results have promised its application in disease therapy. This review aims to summarize the current knowledge in this area and provide a roadmap that may eventually launch RNAi from the research bench to the medicine chest.

RNA interference (RNAi) is an exciting technology with applications in basic research, in elucidation and validation of drug targets, and as a direct therapeutic. In mammalian settings, it is based on the introduction or expression of small interfering RNA (siRNA) that guide the cleavage of a complementary target messenger RNA. While siRNA certainly directs specific silencing of genes in mammalian cells, longer RNA typically used to silence genes in other organisms potently activate mammalian cell defence mechanisms leading to a non-specific halt in translation, to activation of transcription and often, to cell death. Recent research has revealed that siRNA in certain settings can also activate these RNA-responsive pathways. With the recent advances in RNAi technology and its first forays into the in vivo setting now coming to light, it is pertinent to review the cellular response to ribonucleic acids typically used in RNAi methods.

SID-1 is a transmembrane protein that mediates systemic RNA interference in Caenorhabditis elegans. Here we show that the mammalian SID-1 homologue FLJ20174 localizes to the cell membrane of human cells and enhances their uptake of small interfering RNA (siRNA), resulting in increased siRNA-mediated gene silencing efficacy. This is the first demonstration to show that overexpression of a membrane protein enhances siRNA internalization in mammalian cells. This observation raises the possibility of enhancing the efficacy of RNA interference.

RNA interference (RNAi) describes an epigenetic gene silencing reaction by which gene-specific double-stranded RNA acts as a trigger to induce the ribonucleolytic degradation of homologous transcripts. RNAi in African trypanosomes has been shown to be involved in regulating the transcript abundance of retroposons, and the process currently represents the method of choice in gene function studies of the parasite. However, little is known concerning the mechanistic and structural aspects of the processing reaction. This is in part due to the absence of a trypanosome-specific RNAi in vitro system. Here we demonstrate that both the Dicer and the RNA-induced silencing complex steps of the RNAi reaction pathway can be monitored in vitro using cell-free trypanosome extracts. The two in vitro activities and the generated small interfering RNAs (siRNAs) are characterized by features known from other organisms, and we demonstrate that chemically as well as enzymatically synthesized siRNAs are functional in the parasite. Thus, the transfection of synthetic siRNAs can be used to rapidly monitor gene knockdown phenotypes in Trypanosoma brucei, which should be helpful in genome-wide, RNAi-based screening experiments.

RNA silencing pathways convert the sequence information in long RNA, typically double-stranded RNA, into ∼21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs provide specificity to protein effector complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. Here, we review our current understanding of how small RNAs are produced, how they are loaded into protein complexes, and how they repress gene expression.

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression [1]. Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins [2]. Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility [3, 4]. Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3 . Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase β nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2′-5′ oligoadenylate synthetase (OAS), and yeast Trf4p [5]. These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.

RNA interference (RNAi) provides a powerful method for gene silencing in eukaryotic cells. We report on an RNAi technique-induced Pit-1 gene silencing in a GH-secreting cell line MtT/S using duplexes of 21-nt RNAs. By immunocytochemical analysis, the decrease in Pit-1 immunoreactivity was observed in a fraction of siRNA transfected MtT/S cells, while actin and tubulin showed no change in expression following transfection. Moreover, the double staining of Pit-1 and GH indicated that GH production was reduced only in MtT/S cells in which Pit-1 immunoreactivity was decreased by transfection. These results suggested that functional specific knock-down of Pit-1 was induced by transfection of siRNA. Together with the defects of somatotropic lineage and hypoplastic phenotypes in Snell dwarf mice and the transfection experiments using Pit-1 antisense oligonucleotides, our experiments support the theory that Pit-1 plays a crucial role in GH gene expression in individual cells.

There are mainly two types of short RNAs that target complementary messengers in animals: small interfering RNAs and micro-RNAs. Both are produced by the cleavage of double-stranded RNA precursors by Dicer, a member of the Rnase III family of double-stranded specific endonucleases, and both guide the RNA-induced silencing complex to cleave specifically RNAs sharing sequence identity with them. In designing a particular RNA interference (RNAi), it is important to identify the sense/antisense combination that provides the most potent suppression of the target mRNA, and several rules have been established to give >90% gene expression inhibition. RNAi technology can be directed against cancer using a variety of strategies. These include the inhibition of overexpressed oncogenes, blocking cell division by interfering with cyclin E and related genes or promoting apoptosis by suppressing antiapoptotic genes. RNAi against multidrug resistance genes or chemoresistance targets may also provide useful cancer treatments. Studies investigating these approaches in preclinical models are also reviewed.