RNA Gene Silencing Research Articles

Mitotic Cell Shape - RNA Interference Screening for Genes Involved in Mechanics using Atomic Force Microscopy

Cell division in animal cells requires major changes in the cytoskeletal arrangement to achieve accurate positioning of the mitotic spindle and subsequent correct chromosome segregation. To this end, cells undergo drastic shape changes in a process termed mitotic cell rounding. The mechanisms controlling this process are not fully understood. Using an atomic force microscopy (AFM)-based assay that quantifies cell mechanical parameters in conjunction with RNAi gene silencing, we screened almost 1000 genes for their role in mitotic cell mechanics. We find ∼5 % of the genes screened to strongly influence mitotic cell mechanics and provide results from experiments further investigating the role of a subset of these genes formerly not known to be involved in this process. We believe that mechanical phenotyping like presented in this study can lead to new and quantitative insights into the way cells regulate and maintain their shape and mechanical integrity.

Short Hairpin RNA Gene Silencing of Prolyl Hydroxylase-2 with a Minicircle Vector Improves Neovascularization of Hindlimb Ischemia

In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.

Inhibitory effect of chemokine receptor-6-small interfering RNA on human HCCLM6 cell line subcutaneously implanted tumor in nude mice

Objective To investigate the inhibitory effect of chemokine receptor-6 (CCR6)-small interfering RNA (siRNA) gene silencing on human HCCLM6 cells subcutaneously implanted tumor in nude mice.Methods The siRNA targeting CCR6 gene was designed and stably-transfected HCCLM6 cells were subcutaneously implanted into nude mice to established a transplanted tumor model of human hepatocellular carcinoma (HCC).Eighteen mice were divided randomly into three groups (Mock group,CCR6-Scrambled group,CCR6-siRNA group) (n =6 each).Five weeks after implantation,all mice were sacrificed.Tumor growth was calculated by measuring the long/short diameters and the tumor growth curve was delineated.The tumors were weighed and the inhibition rate (IR) obtained.In addition,the metastatic nodules of the liver and lung were evaluated.Results The tumor volume was (843.92 ± 291.08),(809.08 ± 263.74),and (314.17 ±93.42) mm3 in Mock,CCR6-Scrambled and CCR6-siRNA groups respectively.The tumor growth was significantly inhibited in CCR6-siRNA group (P ＜ 0.05).The tumor weight was (1.55 ±0.38),(1.43±0.29),and (0.71 ±0.22) g in Mock,CCR6-Scrambled and CCR6-siRNA groups respectively.The tumor weight in CCR6-siRNA group was significantly reduced (P ＜ 0.05) and the corresponding IR was 52.35％.As compared with Mock and CCR6-Scrambled groups,the metastatic nodules of the liver and lung in CCR6-siRNA group were significantly reduced (P ＜ 0.05).Conclusion CCR6-siRNA could effectively inhibit the growth and metastasis of subcutaneously implanted HCC. Key words: Carcinoma, hepatocellular; Chemokine receptor-6 ; Small interfering RNA

Global Gene Expression and Focused Knockout Analysis Reveals Genes Associated with Fungal Fruiting Body Development in Neurospora crassa

Fungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa.

An RNA-Seq Transcriptome Analysis of Histone Modifiers and RNA Silencing Genes in Soybean during Floral Initiation Process

Epigenetics has been recognised to play vital roles in many plant developmental processes, including floral initiation through the epigenetic regulation of gene expression. The histone modifying proteins that mediate these modifications involve the SET domain-containing histone methyltransferases, JmjC domain-containing demethylase, acetylases and deacetylases. In addition, RNA interference (RNAi)-associated genes are also involved in epigenetic regulation via RNA-directed DNA methylation and post-transcriptional gene silencing. Soybean, a major crop legume, requires a short day to induce flowering. How histone modifications regulate the plant response to external cues that initiate flowering is still largely unknown. Here, we used RNA-seq to address the dynamics of transcripts that are potentially involved in the epigenetic programming and RNAi mediated gene silencing during the floral initiation of soybean. Soybean is a paleopolyploid that has been subjected to at least two rounds of whole genome duplication events. We report that the expanded genomic repertoire of histone modifiers and RNA silencing genes in soybean includes 14 histone acetyltransferases, 24 histone deacetylases, 47 histone methyltransferases, 15 protein arginine methyltransferases, 24 JmjC domain-containing demethylases and 47 RNAi-associated genes. To investigate the role of these histone modifiers and RNA silencing genes during floral initiation, we compared the transcriptional dynamics of the leaf and shoot apical meristem at different time points after a short-day treatment. Our data reveal that the extensive activation of genes that are usually involved in the epigenetic programming and RNAi gene silencing in the soybean shoot apical meristem are reprogrammed for floral development following an exposure to inductive conditions.

GW24-e3668 TXNIP mediates NLRP3 inflammasome activation in cardiac microvascular endothelial cells as a novel mechanism in myocardial ischaemia/reperfusion injury

ObjectivesThe present study sought to determine the role of the NLRP3 inflammasome in MI/R injury and to investigate further the signalling pathways that lead to the activation of NLRP3 inflammasome...

Abstract B39: Methylation of BRCA2 gene promoter CpG units through ZAR2-dependent DNMT1 recruitment

Abstract BRCA2 gene expression is stringently regulated during the cell cycle. BRCA2 expression is proportional to the rate of cell proliferation. Looking at the human BRCA2 gene minimal promoter sequence we found that it has over 45 CpG units which could be potentially methylated regulating the recruitment of different transcription factors affecting BRCA2 gene expression. We discovered that BRCA2 gene promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter (for BRCA2). ZAR2 is a C4-type zinc finger-containing transcription factor that regulates BRCA2 in a cell cycle dependent manner. We found that ZAR2 enters the nucleus of the G0/G1 cells binds to the promoter of BRCA2 and silences the BRCA2 gene expression. ZAR2 has a dsRNA binding domain. We report here that BRCA2 gene promoter, through its bidirectional activity, produces two partially overlapping transcripts (BRCA2 and ZAR2) in the breast cells forming a 111 bp RNA duplex. We postulate that endogenous transcriptional gene silencing RNAs (tgsRNAs) against BRCA2 gene promoter are thus formed from the overlapping RNA duplex in the cell nucleus. These tgsRNAs in turn contribute towards promoter methylation. Our data suggest that ZAR2 upon binding to the tgsRNAs binds to specific locations of BRCA2 gene promoter and recruits DNMT1 which in turn perform CpG methylation. This study will contribute towards the understanding of the epigenetic regulation of BRCA2 gene expression in different cell cycle stages as well as in sporadic cases of breast and other cancer. Supported in part by the DOD-CDMRP IDEA Grants BC990678 and BC050641 to GC and MeTRC pilot project grant 1U54RR026140-01 to SM Citation Format: Smita Misra, Mukul K. Mittal, Sheetal V. Khedkar, Gautam Chaudhuri. Methylation of BRCA2 gene promoter CpG units through ZAR2-dependent DNMT1 recruitment. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B39.

Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1

With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host–virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, like higher plants, may employ certain RNA silencing pathways to defend itself against virus infection. To our knowledge this is the first examination of RNA silencing genes in algae beyond core proteins, and the first analysis of their transcription during virus infection.

Testing insecticidal activity of novel chemically synthesized siRNA against Plutella xylostella under laboratory and field conditions.

BackgroundOver the last 60 years, synthetic chemical pesticides have served as a main tactic in the field of crop protection, but their availability is now declining as a result of the development of insect resistance. Therefore, alternative pest management agents are needed. However, the demonstration of RNAi gene silencing in insects and its successful usage in disrupting the expression of vital genes opened a door to the development of a variety of novel, environmentally sound approaches for insect pest management.Methodology/Principal FindingsSix small interfering RNAs (siRNAs) were chemically synthesized and modified according to the cDNA sequence of P. xylostella acetylcholine esterase genes AChE1 and AChE2. All of them were formulated and used in insecticide activity screening against P. xylostella. Bioassay data suggested that Si-ace1_003 and Si-ace2_001 at a concentration of 3 µg cm−2 displayed the best insecticidal activity with 73.7% and 89.0%, mortality, respectively. Additional bioassays were used to obtain the acute lethal concentrations of LC50 and LC90 for Si-ace2_001, which were 53.66 µg/ml and 759.71 µg/ml, respectively. Quantitative Real-time PCR was used to confirm silencing and detected that the transcript levels of P. xylostella AChE2 (PxAChE2) were reduced by 5.7-fold compared to the control group. Consequently, AChE activity was also reduced by 1.7-fold. Finally, effects of the siRNAs on treated plants of Brassica oleracea and Brassica alboglabra were investigated with different siRNA doses. Our results showed that Si-ace2_001 had no negative effects on plant morphology, color and growth of vein under our experimental conditions.ConclusionsThe most important finding of this study is the discovery that chemically synthesized and modified siRNA corresponding to P. xylostella AChE genes cause significant mortality of the insect both under laboratory and field conditions, which provides a novel strategy to control P. xylostella and to develop bio-pesticides based on the RNA interference technology.

Evaluation of Biodegradable Peptide Carriers for Light-Directed Targeting

A promising strategy for increased intracellular delivery of nucleic acids with the benefit for targeting is photochemical internalization (PCI). PCI relies on the use of a photosensitizing compound that photochemically destroys membranes in the endocytic pathway after illumination, resulting in cytosolic transfer of endosomal content. PCI technology combined with biodegradable polyamino acid carriers and nucleic acids delivers effective targeting and improved biosafety. In an in vitro model system, we have evaluated various poly-l-lysine (PLL), poly-l-histidine (PLH), and poly-l-arginine (PLA) formulations for light-directed small interference RNA (siRNA) gene silencing and messenger RNA (mRNA) delivery. We find that PLA formulations are suitable as siRNA and mRNA carriers in a strictly light-directed manner.

Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models

Nicotinamide adenine dinucleotide (NAD)+, a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD+ expression has been linked with a decrease in beta-amyloid (Aβ) toxicity in Alzheimer's disease (AD). Nicotinamide riboside (NR) is a NAD+ precursor, it promotes peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α expression in the brain. Evidence has shown that PGC-1α is a crucial regulator of Aβ generation because it affects β-secretase (BACE1) degradation. In this study we tested the hypothesis that NR treatment in an AD mouse model could attenuate Aβ toxicity through the activation of PGC-1α-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysiological recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD+ in the cerebral cortex; (2) application of NR to hippocampal slices (10 μM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1 degradation was prevented by PGC-1α-shRNA gene silencing; and (4) NR treatment and PGC-1α overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, in part by promoting PGC-1α-mediated BACE1 ubiquitination and degradation, thus preventing Aβ production in the brain.

Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing

Despite prolonged and intensive application, combined antiretroviral therapy cannot eradicate human immunodeficiency virus (HIV)-1 because it is harbored as a latent infection, surviving for long periods of time. Alternative approaches are required to overcome the limitations of current therapy. We have been developing a short interfering RNA (siRNA) gene silencing approach. Certain siRNAs targeting promoter regions of genes induce transcriptional gene silencing. We previously reported substantial transcriptional gene silencing of HIV-1 replication by an siRNA targeting the HIV-1 promoter in vitro. In this study, we show that this siRNA, expressed as a short hairpin RNA (shRNA) (shPromA-JRFL) delivered by lentiviral transduction of human peripheral blood mononuclear cells (PBMCs), which are then used to reconstitute NOJ mice, is able to inhibit HIV-1 replication in vivo, whereas a three-base mismatched variant (shPromA-M2) does not. In shPromA-JRFL–treated mice, HIV-1 RNA in serum is significantly reduced, and the ratio of CD4+/CD8+ T cells is significantly elevated. Expression levels of the antisense RNA strand inversely correlates with HIV-1 RNA in serum. The silenced HIV-1 can be reactivated by T-cell activation in ex vivo cultures. HIV-1 suppression is not due to offtarget effects of shPromA-JRFL. These data provide “proof-of principle” that an shRNA targeting the HIV-1 promoter is able to suppress HIV-1 replication in vivo.

RKN Lethal DB: A database for the identification of Root Knot Nematode (Meloidogyne spp.) candidate lethal genes.

Root Knot nematode (RKN; Meloidogyne spp.) is one of the most devastating parasites that infect the roots of hundreds of plant species. RKN cannot live independently from their hosts and are the biggest contributors to the loss of the world's primary foods. RNAi gene silencing studies have demonstrated that there are fewer galls and galls are smaller when RNAi constructs targeted to silence certain RKN genes are expressed in plant roots. We conducted a comparative genomics analysis, comparing RKN genes of six species: Meloidogyne Arenaria, Meloidogyne Chitwoodi, Meloidogyne Hapla, Meloidogyne Incognita, Meloidogyne Javanica, and Meloidogyne Paranaensis to that of the free living nematode Caenorhabditis elegans, to identify candidate genes that will be lethal to RKN when silenced or mutated. Our analysis yielded a number of such candidate lethal genes in RKN, some of which have been tested and proven to be effective in soybean roots. A web based database was built to house and allow scientists to search the data. This database will be useful to scientists seeking to identify candidate genes as targets for gene silencing to confer resistance in plants to RKN.AvailabilityThe database can be accessed from http://bioinformatics.towson.edu/RKN/

SnapShot: Endogenous RNA Pathways

Endogenous RNAi (endoRNAi) identifies a heterologous but interrelated array of small RNA-mediated phenomena triggered by and directing the silencing of endogenous sequences. EndoRNAi mechanisms target aberrant transcripts and repetitive and mobile RNA sequences, as well as bona fide messenger RNAs. In most endoRNAi mecha-nisms, the underlying molecular events remain poorly defined but appear to be almost as diverse as the targeted loci. A great variety of triggers can initiate endoRNAi in differ-ent cell types and species, each resulting in specific modes of small RNA biogenesis, response amplification, and gene silencing. In spite of such diversity, redundant themes prevail among the endoRNAi mechanisms. Our objective was to capture most of those themes. Note that, although it would conceptually fit within the continuum of endoRNAi mechanisms, canonical microRNA-mediated silencing is usually considered separately. This specific pathway has been extensively reviewed and is therefore not included in this SnapShot.In certain species of yeasts, protozoa (such as

Cajal Bodies and Plant RNA Metabolism

Intracellular location of components associated with RNA metabolism is a key feature of gene expression regulation, although it is often neglected in favor of more direct factors influencing expression. A number of nuclear and subnuclear compartments exist, each carrying out specific functions. This article focuses on Cajal bodies, a dynamic body composed of numerous proteins and small RNAs. Cajal bodies, or CBs, are universal entities found in both the nucleolus and nucleoplasm of most living cells. Their function relates to RNA metabolism, including transcription, splicing, stable RNA modification, small RNA biogenesis, gene silencing, nonsense-mediated decay and telomere maintenance. It is generally thought that CBs form docking stations for each of these functions so that the individual processes proceed quickly and in an organized manner. A brief introduction on what is known about the function and composition of CBs in animal cells is provided, followed by highlights of the latest research on CBs in plants.

The molecular architecture of human Dicer

Dicer is a multi-domain enzyme that generates small RNAs for gene silencing in eukaryotes. Current understanding of Dicer structure is restricted to simple forms of the enzyme, while that of the large and complex Dicer, widespread in eukarya, is unknown. Here, we describe a novel domain localization strategy developed to determine the structure of human Dicer by electron microscopy. A rearrangement of the nuclease core, compared to the archetypal Giardia Dicer, explains how metazoan Dicers generate 21–23 nucleotide products. The helicase domains form a clamp-like structure adjacent to the RNase III active site, facilitating recognition of pre-miRNA loops or translocation on long dsRNAs. Drosophila Dicer-2 displays similar features, revealing that the three-dimensional architecture is conserved. These results illuminate the structural basis for small RNA production in eukaryotes and provide a versatile new tool for determining structures of large molecular machines.

Anopheles gambiae Antiviral Immune Response to Systemic O'nyong-nyong Infection

BackgroundMosquito-borne viral diseases cause significant burden in much of the developing world. Although host-virus interactions have been studied extensively in the vertebrate host, little is known about mosquito responses to viral infection. In contrast to mosquitoes of the Aedes and Culex genera, Anopheles gambiae, the principal vector of human malaria, naturally transmits very few arboviruses, the most important of which is O'nyong-nyong virus (ONNV). Here we have investigated the A. gambiae immune response to systemic ONNV infection using forward and reverse genetic approaches.Methodology/Principal FindingsWe have used DNA microarrays to profile the transcriptional response of A. gambiae inoculated with ONNV and investigate the antiviral function of candidate genes through RNAi gene silencing assays. Our results demonstrate that A. gambiae responses to systemic viral infection involve genes covering all aspects of innate immunity including pathogen recognition, modulation of immune signalling, complement-mediated lysis/opsonisation and other immune effector mechanisms. Patterns of transcriptional regulation and co-infections of A. gambiae with ONNV and the rodent malaria parasite Plasmodium berghei suggest that hemolymph immune responses to viral infection are diverted away from melanisation. We show that four viral responsive genes encoding two putative recognition receptors, a galectin and an MD2-like receptor, and two effector lysozymes, function in limiting viral load.Conclusions/SignificanceThis study is the first step in elucidating the antiviral mechanisms of A. gambiae mosquitoes, and has revealed interesting differences between A. gambiae and other invertebrates. Our data suggest that mechanisms employed by A. gambiae are distinct from described invertebrate antiviral immunity to date, and involve the complement-like branch of the humoral immune response, supressing the melanisation response that is prominent in anti-parasitic immunity. The antiviral immune response in A. gambiae is thus composed of some key conserved mechanisms to target viral infection such as RNAi but includes other diverse and possibly species-specific mechanisms.

RET PLCγ Phosphotyrosine Binding Domain Regulates Ca2+ Signaling and Neocortical Neuronal Migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca2+) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.

Molecular Mechanisms of RNA-Triggered Gene Silencing Machineries

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.

Characterization of an HIV-targeted transcriptional gene-silencing RNA in primary cells.

Small antisense RNAs targeted to the HIV-1 promoter have been shown to remodel the surrounding chromatin to a state unfavorable for transcriptional activation, yet transcriptional gene silencing (TGS) of HIV-1 has, to date, not been shown in primary human cells. We demonstrate here that TGS can reduce viral transcription in primary human CD4(+) T cells; however, increasing viral burden results in the loss of this antiviral effect. This observation suggests a critical level at which viral RNA can dilute out effective targeting by TGS-based RNAs. Furthermore, studies into off-target effects have identified a potential interaction between the small nucleolar RNA pathway and the TGS-based antisense RNA, resulting in activation of p53. Although not overtly toxic to primary cells, this represents a novel interaction between antisense RNAs and a cellular pathway that should be considered when pursuing small antisense RNA-based therapeutics.