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Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequence-specific post-transcriptional regulators of gene expression. Although major components of the RNA interference (RNAi) pathway have been identified, regulatory mechanisms for this pathway remain largely unknown. Here we demonstrate that the RNAi pathway can be modulated intracellularly by small molecules. We have developed a cell-based assay to monitor the activity of the RNAi pathway and find that the small-molecule enoxacin (Penetrex) enhances siRNA-mediated mRNA degradation and promotes the biogenesis of endogenous miRNAs. We show that this RNAi-enhancing activity depends on the trans-activation-responsive region RNA-binding protein. Our results provide a proof-of-principle demonstration that small molecules can be used to modulate the activity of the RNAi pathway. RNAi enhancers may be useful in the development of research tools and therapeutics.

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.

The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host-virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics.

Small interfering RNAs (siRNAs) and genome-encoded microRNAs (miRNAs) silence genes via complementary interactions with mRNAs. With thousands of miRNA genes identified and genome sequences of diverse eukaryotes available for comparison, the opportunity emerges for insights into origin and evolution of RNA interference (RNAi). The miRNA repertoires of plants and animals appear to have evolved independently. However, conservation of the key proteins involved in RNAi suggests that the last common ancestor of modern eukaryotes possessed siRNA-based mechanisms. Prokaryotes have a RNAi-like defense system that is functionally analogous but not homologous to eukaryotic RNAi. The protein machinery of eukaryotic RNAi seems to have been pieced together from ancestral proteins of archaeal, bacterial and phage origins that are involved in DNA repair and RNA-processing pathways.

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.

Background

RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems.

Results

Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate.

Conclusion

This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries.

The phenomenon that is known as RNA mediated interference (RNAi) was first observed in the nematode C. elegans. The application of RNAi has now been widely disseminated and the mechanisms underlying the pathway have been uncovered using both genetics and biochemistry. In the worm, it has been demonstrated that RNAi is easily adapted to high throughput analysis and screening protocols. Hence, given the availability of whole genome sequences, RNAi has been used extensively as a tool for annotating gene function. Genetic screens performed with C. elegans have also led to the identification of genes that are essential for RNAi or that modulate the RNAi process. The identification of such genes has made it possible to manipulate and enhance the RNAi response. Moreover, many of the genes identified in C. elegans have been conserved in other organisms. Thus, opportunities are available for researchers to take advantage of the insights gained from the worm and apply them to their own systems in order to improve the efficiency and potency of the RNAi response.

RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpesviral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.

The discovery of RNA interference (RNAi) in C. elegans and in plants has revolutionized current approaches to biology and medicine. RNAi silences genes in a sequence-specific manner through the actions of small pieces of double-stranded RNAs (siRNAs and miRNAs). RNAi has been found as a widespread natural phenomenon in eukaryotic cells and is also being used as a powerful experimental tool to explore gene function. Most importantly, it has many potential therapeutic applications. Viral gene-specific siRNAs are theoretically very promising antiviral inhibitors and have been examined in a broad range of medically important viruses. However, many RNA viruses escape RNAi-mediated suppression by counteracting the RNAi machinery through mutation of the targeted region, by encoding viral suppressors, or both. DNA viruses also counteract the RNAi machinery, preferentially using viral suppressors. Cellular factors may also contribute to RNAi resistance; ADAR1 was the first cellular factor found to be responsible for editing-mediated RNAi resistance. Because siRNAs can be used as potent small-molecule inhibitors of any cellular gene, the best way for a cell to maintain expression of essential genes for its long-term survival is to develop a program to resist the detrimental effects of RNAi.

Small non-coding RNAs such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA) exist in almost all kingdoms of organisms and have recently emerged as master regulators of gene expression to affect a diverse range of important biological processes. They exert their functions largely through two related but opposing mechanisms: RNA interference (RNAi) mediated by siRNA, miRNA and piRNA, and RNA activation (RNAa) mediated by small activating RNA (saRNA) and miRNA, leading to silencing and overexpression of target genes respectively. Dysregulation of these mechanisms have been implicated in a variety of human diseases including urological and andrological diseases. Importantly, both mechanisms can be readily harnessed for therapeutic purposes for a variety of diseases by using small RNA molecules as the “ribodrug”. In this review, we highlight recent advances in the applications of small RNA as therapeutics for urological cancer, male infertile and erectile dysfunction.

RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.

RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.

Author Summary

RNA interference (RNAi) has become a widely used approach for silencing genes of interest. This tool is possible because endogenous RNA silencing pathways exist broadly across organisms, including humans, worms, and plants. The general RNAi pathway utilizes small ∼21-nucleotide RNAs to target specific protein-coding genes through base-pairing interactions. Since RNAs from exogenous sources require some of the same factors as endogenous small RNAs to silence gene expression, there can be competition between the pathways. Thus, perturbations in the endogenous RNAi pathway can result in enhanced silencing efficiency by exogenous small RNAs. MicroRNAs (miRNAs) comprise another endogenous small RNA pathway, but their biogenesis and mechanism of gene silencing are distinct in many ways from RNAi pathways. Here we show that a family of miRNAs regulates the effectiveness of RNAi in Caenorhabditis elegans. Loss of mir-35-41 results in enhanced RNAi by exogenous RNAs and reduced silencing of endogenous RNAi targets. The embryonic miR-35-41 miRNAs regulate the sensitivity to RNAi through lin-35/Rb, a homolog of the human Retinoblastoma tumor suppressor gene previously shown to regulate RNAi effectiveness in C. elegans. Additionally, we show that this sensitivity can be passed on to the next generation of worms, demonstrating a far-reaching effect of the miR-35-41 miRNAs on gene regulation by other small RNA pathways.

Background

RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA.

Results

In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing.

Conclusion

HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency.

Background

High-throughput RNA interference (RNAi) screens have been used to find genes that, when silenced, result in sensitivity to certain chemotherapy drugs. Researchers therefore can further identify drug-sensitive targets and novel drug combinations that sensitize cancer cells to chemotherapeutic drugs. Considerable uncertainty exists about the efficiency and accuracy of statistical approaches used for RNAi hit selection in drug sensitivity studies. Researchers require statistical methods suitable for analyzing high-throughput RNAi screening data that will reduce false-positive and false-negative rates.

Results

In this study, we carried out a simulation study to evaluate four types of statistical approaches (fold-change/ratio, parametric tests/statistics, sensitivity index, and linear models) with different scenarios of RNAi screenings for drug sensitivity studies. With the simulated datasets, the linear model resulted in significantly lower false-negative and false-positive rates. Based on the results of the simulation study, we then make recommendations of statistical analysis methods for high-throughput RNAi screening data in different scenarios. We assessed promising methods using real data from a loss-of-function RNAi screen to identify hits that modulate paclitaxel sensitivity in breast cancer cells. High-confidence hits with specific inhibitors were further analyzed for their ability to inhibit breast cancer cell growth. Our analysis identified a number of gene targets with inhibitors known to enhance paclitaxel sensitivity, suggesting other genes identified may merit further investigation.

Conclusions

RNAi screening can identify druggable targets and novel drug combinations that can sensitize cancer cells to chemotherapeutic drugs. However, applying an inappropriate statistical method or model to the RNAi screening data will result in decreased power to detect the true hits and increase false positive and false negative rates, leading researchers to draw incorrect conclusions. In this paper, we make recommendations to enable more objective selection of statistical analysis methods for high-throughput RNAi screening data.

Systems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline.

Background

RNA interference (RNAi) is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs).

Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes.

Results

As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system.

Conclusions

We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.

RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species.

Background

MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs (ncRNAs) which play important roles in eukaryotic gene regulation. miRNA biogenesis and activation is a complex process involving multiple protein catalysts and involves the large macromolecular RNAi Silencing Complex or RISC. While phylogenetic analyses of miRNA genes have been previously published, the evolution of miRNA biogenesis itself has been little studied. In order to better understand the origin of miRNA processing in animals and plants, we determined the phyletic occurrences and evolutionary relationships of four major miRNA pathway protein components; Dicer, Argonaute, RISC RNA-binding proteins, and Exportin-5.

Results

Phylogenetic analyses show that all four miRNA pathway proteins were derived from large multiple protein families. As an example, vertebrate and invertebrate Argonaute (Ago) proteins diverged from a larger family of PIWI/Argonaute proteins found throughout eukaryotes. Further gene duplications among vertebrates after the evolution of chordates from urochordates but prior to the emergence of fishes lead to the evolution of four Ago paralogues. Invertebrate RISC RNA-binding proteins R2D2 and Loquacious are related to other RNA-binding protein families such as Staufens as well as vertebrate-specific TAR (HIV trans-activator RNA) RNA-binding protein (TRBP) and protein kinase R-activating protein (PACT). Export of small RNAs from the nucleus, including miRNA, is facilitated by three closely related karyopherin-related nuclear transporters, Exportin-5, Exportin-1 and Exportin-T. While all three exportins have direct orthologues in deutrostomes, missing exportins in arthropods (Exportin-T) and nematodes (Exportin-5) are likely compensated by dual specificities of one of the other exportin paralogues.

Conclusion

Co-opting particular isoforms from large, diverse protein families seems to be a common theme in the evolution of miRNA biogenesis. Human miRNA biogenesis proteins have direct, orthologues in cold-blooded fishes and, in some cases, urochordates and deutrostomes. However, lineage specific expansions of Dicer in plants and invertebrates as well as Argonaute and RNA-binding proteins in vertebrates suggests that novel ncRNA regulatory mechanisms can evolve in relatively short evolutionary timeframes. The occurrence of multiple homologues to RNA-binding and Argonaute/PIWI proteins also suggests the possible existence of further pathways for additional types of ncRNAs.

In recent years RNA interference (RNAi) has rapidly become the most widely used tool for gene knockdown due to its high specificity and potency. RNAi is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. In the past decade, hundreds of molecular targets have been identified for their roles in pain modulation. But most molecular targets are not readily druggable with small molecules. RNAi represents a therapeutic approach applicable to these non-druggable targets. There is a rapid increase in the number of studies that use small interfering RNAs (siRNAs) to validate new targets for pain regulation. In this review, we will discuss these pain-related RNAi studies (Table 1). We will also compare the advantages and disadvantages of RNAi with antisense knockdown (Table 2), because antisense oligodeoxynucleotides have been extensively used for target validation in pain research. Although in vivo delivery of siRNA remains to be a challenge, RNAi has a great potential to become a major therapeutic tool for pain management.

Summary

Background

miRNAs are an abundant class of small, endogenous regulatory RNAs. Although it is now appreciated that miRNAs are involved in a broad range of biological processes, relatively little is known about the actual mechanism by which miRNAs down-regulate target gene expression. An exploration of what protein co-factors are necessary for a miRNA to down-regulate a target gene should reveal more fully the molecular mechanisms by which miRNAs are processed, trafficked, and regulate their target genes.

Results

A weak allele of the C. elegans miRNA gene let-7 was used as a sensitized genetic background for a whole-genome RNAi screen to detect miRNA pathway genes, and 213 candidate miRNA pathway genes were identified. About 2/3 of the 61 candidates with the strongest phenotype were validated through genetic tests examining the dependence of the let-7 phenotype on target genes known to function in the let-7 pathway. Biochemical tests for let-7 miRNA production place the function of nearly all of these new miRNA pathway genes downstream of let-7 expression and processing. By monitoring the down-regulation of the protein product of the lin-14 mRNA, which is the target of the lin-4 miRNA, we have identified 19 general miRNA pathway genes.

Conclusions

The 213 candidate miRNA pathway genes identified could act at steps that produce and traffic miRNAs or in downstream steps that detect miRNA::mRNA duplexes to regulate mRNA translation. The 19 validated general miRNA pathway genes are good candidates for genes that may define protein cofactors for sorting or targeting miRNA::mRNA duplexes, or recognizing the miRNA basepaired to the target mRNA to down-regulate translation.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through the RNA interference (RNAi) pathway and by inhibition of mRNA translation. miRNAs first made their appearance in the auditory and vestibular systems in 2005, with the discovery of a triad of hair cell-specific miRNAs later found to be involved in both human and mouse deafness. Since then, miRNAs have been implicated in other medical conditions related to these systems, such as cholesteatomas, vestibular schwannomas and otitis media. Due to the limitations in studying miRNAs and their targets derived from human inner ears, animal models are vital in this field of research. Therefore their role in inner ear development and function has been demonstrated by studies in zebrafish and mice. Transcriptomic and proteomic approaches have been undertaken to identify miRNAs and their targets. Finally, it has been suggested that miRNAs may be used in the future in regeneration of inner ear hair cells and ultimately play a role in therapeutics.

RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.