Post-translational Gene Silencing Research Articles

RNA Interference Past and Future Applications in Plants.

Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement.

Transcriptional Landscape of Small Non-coding RNAs in Somatic and Gonadal Tissues of Brachyuran Crabs (Genus Eriocheir and Portunus)

Small non-coding RNAs (sncRNAs) play important roles in transcriptional and post-translational gene silencing in animals. However, the sncRNAs were not well studied in Brachyuran crabs, especially for the PIWI-interacting RNAs (piRNAs), which suppress transposon activity to maintain the germ lines genomic integrity. To unravel the sncRNAs expression profiling and genomic function in Brachyuran crabs, we generated a comprehensive small RNA dataset by small RNA sequencing (sRNA-seq) of three crabs. Abundant known and novel miRNAs were identified and characterized in these crabs. Among these miRNAs, 12 miRNAs were detected in all crabs, and another 41 miRNAs were detected in both of E. sinensis and E. japonica. In addition, abundant piRNA reads with a significant ping-pong signal were discovered in the ovaries and testis of Brachyuran crabs, which might associate with transposon silencing in germ lines. Our dataset would serve as important reference data for future genomic and genetic studies on small RNAs in these crabs and related species.

A Major Facilitator Superfamily Peptide Transporter From Fusarium oxysporum Influences Bioethanol Production From Lignocellulosic Material.

Fusarium oxysporum is a leading microbial agent in the emerging consolidated bioprocessing (CBP) industry owing to its capability to infiltrate the plant’s lignin barrier and degrade complex carbohydrates to value-added chemicals such as bioethanol in a single step. Membrane transport of nutrients is a key factor in successful microbial colonization of host tissue. This study assessed the impact of a peptide transporter on F. oxysporum’s ability to convert lignocellulosic straw to ethanol. We characterized a novel F. oxysporum peptide transporter (FoPTR2) of the dipeptide/tripeptide transporter (PTR) class. FoPTR2 represents a novel transporter with high homology to the Trichoderma sp. peptide transporters ThPTR2 and TrEST-AO793. Its expression level was highly activated in nitrogen-poor environments, which is a characteristic of PTR class peptide transporters. Overexpression and post-translational gene silencing of the FoPTR2 in F. oxysporum affected the peptide transport capacity and ethanol yielded from a both a wheat straw/bran mix and glucose. Thus, we conclude that it FoPTR2 plays a role in the nutrient acquisition system of F. oxysporum which serves to not only enhance fungal fitness but also CBP efficacy.

Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections.

Several viruses cause pulmonary infections due to their shared tropism with cells of the respiratory tract. These respiratory problems due to viral infection become a public health concern due to rapid transmission through air/aerosols or via direct-indirect contact with infected persons. In addition, the cross-species transmission causes alterations to viral genetic makeup thereby increasing the risk of emergence of pathogens with new and more potent infectivity. With the introduction of effective nucleic acid-based technologies, post translational gene silencing (PTGS) is being increasingly used to silence viral gene targets and has shown promising approach towards management of many viral infections. Since several host factors are also utilized by these viruses during various stages of infection, silencing these host factors can also serve as promising therapeutic tool. Several nucleic acid-based technologies such as short interfering RNAs (siRNA), antisense oligonucleotides, aptamers, deoxyribozymes (DNAzymes), and ribozymes have been studied and used against management of respiratory viruses. These therapeutic nucleic acids can be efficiently delivered through the airways. Studies have also shown efficacy of gene therapy in clinical trials against respiratory syncytial virus (RSV) as well as models of respiratory diseases including severe acute respiratory syndrome (SARS), measles and influenza. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and highlighted the emerging roles of nucleic acids in the management of some of the severe respiratory viral infections. We have also focused on the methods of their delivery and associated challenges.

PsRNATarget: a plant small RNA target analysis server (2017 release).

Plant regulatory small RNAs (sRNAs), which include most microRNAs (miRNAs) and a subset of small interfering RNAs (siRNAs), such as the phased siRNAs (phasiRNAs), play important roles in regulating gene expression. Although generated from genetically distinct biogenesis pathways, these regulatory sRNAs share the same mechanisms for post-translational gene silencing and translational inhibition. psRNATarget was developed to identify plant sRNA targets by (i) analyzing complementary matching between the sRNA sequence and target mRNA sequence using a predefined scoring schema and (ii) by evaluating target site accessibility. This update enhances its analytical performance by developing a new scoring schema that is capable of discovering miRNA–mRNA interactions at higher ‘recall rates’ without significantly increasing total prediction output. The scoring procedure is customizable for the users to search both canonical and non-canonical targets. This update also enables transmitting and analyzing ‘big’ data empowered by (a) the implementation of multi-threading chunked file uploading, which can be paused and resumed, using HTML5 APIs and (b) the allocation of significantly more computing nodes to its back-end Linux cluster. The updated psRNATarget server has clear, compelling and user-friendly interfaces that enhance user experiences and present data clearly and concisely. The psRNATarget is freely available at http://plantgrn.noble.org/psRNATarget/.

Application of RNA interference methodology to investigate and develop SCMV resistance in maize.

Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach.

MYMIV-AC2 protein suppresses hairpin-induced gene silencing in Nicotiana tabacum cv. Xanthi

Construct encoding self-complementary ‘hairpin’ RNA (hpRNA) has been shown as an effective trigger of post-translational gene silencing. Gene silencing can be effectively inhibited by virus-encoded silencing suppressors that have been acquired evolutionarily to surmount silencing and allow infection. Here, we show that AC2, the silencing suppressor protein of Mungbean Yellow Mosaic India Virus (MYMIV), can inhibit the hpRNA-induced gene silencing in planta. Phytoene desaturase (PDS)-silenced transgenic lines were generated using PDS-hairpin gene-silencing construct in Nicotiana. The photobleach phenotype was observed in different T1 and T2 PDS-silenced lines indicating that the hairpin gene-silencing construct can efficiently down-regulate or silence the endogenous gene activity, and that the silencing is stably inherited over generations. The analysis of abundance of the PDS transcripts showed that the degree of silencing ranged from 20 to 70 % in different T0 and T1 transgenic lines. Upon introduction of MYMIV-AC2 in the silenced PDS transgenic lines, by genetic hybridization, the PDS expression was restored in F1 hybrids. The F1 lines (♀PDS X ♂MYMIV-AC2) showed the normal green phenotype instead of the photobleached phenotype indicating that introgression of MYMIV-AC2 in PDS silenced T0 lines, suppresses the hairpin gene-silencing. Our findings suggest that hpRNA vector can efficiently and stably induce gene silencing in plants and that MYMIV-AC2 can inhibit this silencing. This is the first report where we put forward that the dual system of hpRNA and MYMIV-AC2 suppressor can serve as an efficient switch for turning off and turning on gene expression, with potential applications in gene function analysis.

Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

Comprehensive annotation of microRNA expression profiles

BackgroundMicroRNAs (miRNAs) regulate many biological processes by post-translational gene silencing. Analysis of miRNA expression profiles is a reliable method for investigating particular biological processes due to the stability of miRNA and the development of advanced sequencing methods. However, this approach is limited by the broad specificity of miRNAs, which may target several mRNAs.ResultIn this study, we developed a method for comprehensive annotation of miRNA array or deep sequencing data for investigation of cellular biological effects. Using this method, the specific pathways and biological processes involved in Alzheimer’s disease were predicted with high correlation in four independent samples. Furthermore, this method was validated for evaluation of cadmium telluride (CdTe) nanomaterial cytotoxicity. As a result, apoptosis pathways were selected as the top pathways associated with CdTe nanoparticle exposure, which is consistent with previous studies.ConclusionsOur findings contribute to the validation of miRNA microarray or deep sequencing results for early diagnosis of disease and evaluation of the biological safety of new materials and drugs.

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.

Abstract 5040: Aberrant DNA methylation of microRNAs in hepatocellular carcinoma

Abstract microRNAs (miRNAs) are a class of small non-coding RNAs involved in post-translational gene silencing by accelerating the degradation of mRNA and translational inhibition. miRNAs may regulate more than 60% of protein-coding genes’ translation. Previous studies have found that down-regulation of miRNAs is one of the most common features observed in solid tumors, including hepatocellular cancer (HCC). In the current study, we employed a genome-wide approach to test the hypothesis that methylation alteration in miRNA genes is an important mechanism causing deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using the HumanMethylation27 DNA Analysis BeadChips that include 255 CpG sites relevant to 64 miRNA genes. A volcano plot was used to display mean DNA methylation differences of all CpG sites. The hierarchical clustering for the significant CpG sites was performed to differentiate tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites (27 relevant genes) significantly differed in methylation levels, with 53 CpG sites significantly hypermethylated in tumor compared to non-tumor tissues. Among the significant CpG sites, 15 sites have more than 2 fold tumor/non-tumor differences, 17 sites have a difference between tumor and non-tumor tissues of >10%, and 10 sites have both features. PANTHER ontology analysis suggested several enriched biological pathways for these miRNAs, including Wnt signaling, Ras, oxidative stress response, glutamine/glutamate conversion and mannose metabolism. The miRNAs methylation profiles were also compared by viral status (HBV/HCV) and AFB1 exposures. No statistically significant miRNA was identified. Analysis of a panel of candidate miRNAs (miR-196, miR-10a, miR-10b and miR-170) is ongoing in the same HCC tissues to evaluate whether the aberrant DNA methylation significantly correlates with expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5040. doi:1538-7445.AM2012-5040

Apoptosis induced by nonspecific effects of siRNA in human umbilical vein endothelial cell

RNA interference (RNAi) is a powerful tool used to produce post-translational gene silencing in a sequence-dependent manner. However, sequence-independent activation of interferon systems induced by double-stranded RNA can interfere with the data interpretation of RNAi experiments. In this study, we observed cytokine activation and cytotoxicity caused by small interfering RNA (siRNA) in human umbilical vein endothelial cells (HUVECs). Transfection with different sequences of siRNAs (21-nucleotides) induced apoptosis in HUVECs within 24 hours, in a dose-dependent and sequence-independent manner. These effects could not be achieved by the application of transfection agents nor by siRNA only. In HUVECs, the expression of toll-like receptor 3 (TLR3), a receptor for double-stranded RNA, was significantly increased by the transfection with siRNAs regardless of their sequences. The expressions of interleukin 6, interleukin 8, and interferon-β were also up-regulated by siRNA transfection. The activation of RNA-dependent protein kinase (PKR) peaked within 30 min and slowly decreased 4 hours after transfection with siRNA, which was followed by the phosphorylation of eukaryotic initiation factor 2α(eIF2α). These studies suggest that transfection with double-stranded oligonucleotides in HUVECs shows sequence-independent apoptotic effects associated with the upregulation of TLR3 and cytokines, which should be considered when designing experiments using RNAi.

SiRNA-optimized Modifications for Enhanced In Vivo Activity

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data.

2007 In Vitro Biology meeting 2007 meeting of the Society for In Vitro Biology June 9–13, Indianapolis, IN

Toll-like receptors (TLRs) constitute an essential family of pattern recognition molecules that, through the direct recognition of conserved microbial components, initiate inflammatory responses after infection. Phylogenetic evidence suggests that vertebrate TLRs are under strong purifying selection for the maintenance of function. Our lab is focused on a related group of vertebrate TLRs that comprise the TLR2 subfamily. The most closely related members, TLRs 1 and 6, appear to have arisen from a recent gene duplication event and have acquired differential microbial recognition specificity. We have characterized two single nucleotide polymorphisms (SNPs), P315L, and I602S, in human TLR1 that effect receptor function through different mechanisms. The 315L variant is associated with deficient recognition of microbial products whereas the 602S variant is associated with aberrant trafficking of the receptor to the cell surface. It is surprising to note that the 602S allele is associated with a decreased incidence of leprosy suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion. TLR1 I602S exhibits strikingly different allele frequencies among different races suggesting that if strong purifying selection took place, it was restricted by either additional genetic or environmental factors that were geographically constrained.High expression of transgenes is desired for molecular farming. However, transgenes are often subjected to gene silencing pathways in plant cells. Gene silencing may be triggered by the production of aberrant (hairpin) RNA molecule from a complex integration locus in plant genome or by the overexpression of transgene. Excessively transcribed RNA are subject to gene silencing even if they are produced from a single-copy locus. Therefore, we sought to determine how highly a transgene can be expressed before its transcript is subjected to gene silencing. In a previous study, we demonstrated that precise single-copy locus generated by Cre/lox-mediated site-specific integration (SSI) in rice is stably expressed at predictable levels through subsequent generations, and that its expression invariably doubled in homozygous progenies. To further explore the stability of the SSI locus and determine the expression-threshold of rice genomic sites, we generated transformation vectors containing 1–3 copies of 35S-GUS and 35S-GFP transgenes. These vectors were used to develop SSI locus containing 1–3 copies of each transgene in two different varieties of rice, Nipponbare and Taipei 309. SSI lines will be analyzed by PCR and Southern blotting to ascertain the presence of precise integration structures consisting of 1–3 copies of each trans-gene. The precise SSI lines will be subjected to quantitative GUS and GFP assay to determine if gene expression indeed increased with the increase in gene dosage. Molecular and expression data of SSI lines will be presented.Tobacco plants (Nicotiana tabacum L.) were transformed with a construct based on pCAMBIA 2301 containing a “hairpin” inverted repeat of 598 nucleotides derived from the Potato Virus Y (PVY) replicase (NIb gene) of the N strain (Robaglia et al., J. Gen. Virol. 70, 935, 1989). Such constructs confer virus resistance by a post translational gene silencing mechanism. Homozygous (T3) plants were challenged with a range of PVY strains and resistance was measured by symptom expression, ELISA titer, and back inoculation of controls with extracts from resistant plants. The nucleotide homology of PVY strains to the transgene was: WP (99.5%) PVY-NTN (96.3), PVY-H (95.6%), PVY-O (88.9%), strain 52 (88.3%), and local field isolates from tomato (86.8%), and pepper (86.3%). A transgenic tobacco line was immune to the five PVY strains with which the transgene had the greatest homology (WP, NTN, H, O, 52). Infection with the PVY isolates from tomato and pepper, which had the lowest degree of homology with the transgene, caused delayed symptom appearan

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

Recapitulation of Short RNA-Directed Translational Gene Silencing In Vitro

microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.

Viral-based modelling and correction of neurodegenerative diseases by RNA interference

Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.

Gene Disruption by Regulated Short Interfering RNA Expression, Using a Two-Adenovirus System

Specific gene ablation by RNA inference (RNAi) involves the binding of short interfering RNA (siRNA), 21 to 22 nucleotides long, to complementary mRNA sequences, leading to sequence-specific posttranslational gene silencing, thus providing a powerful tool for studying gene function with potential therapeutic applications. Here we describe the development of a two-vector adenovirus system for efficient, tightly controlled hairpin siRNA expression (shRNA). Regulated expression of the shRNA is conferred within an adenoviral vector by a modified RNA polymerase III promoter containing a Tet operator element adjacent to the transcription start site. In the presence of the tetracycline repressor protein (TetR), encoded in a second adenovirus, shRNA expression is repressed. Addition of tetracycline abolishes TetR binding, allowing shRNA transcription to proceed, and leading to reduced mRNA and protein expression. Here we establish the efficacy of this system by delivering siRNA targeted against the transcriptional coactivator p300. Our results show tetracycline-mediated inhibition of p300 mRNA and protein accumulation in the presence of both viruses, but no effect in the absence of antibiotic. Regulated adenoviral shRNA vectors offer the advantages of being able to infect a wide array of replicating and nonreplicating cells and of allowing temporal control of gene silencing.

Small RNAs with a not-so-small role in Arabidopsis

Recent studies in plants and animals have shown that 20–24 bp small-interfering RNAs (siRNAs) are necessary for post-translational gene silencing (PTGS) and RNA interference (RNAi). Beginning with work in Caenorhabditis elegans on the RNAs lin-4 and let-7, it has become clear that RNAs of a similar size, termed microRNAs (miRNAs), can be important for the normal regulation of endogenous genes. Like siRNAs, miRNAs arise from the cleavage of double-stranded RNA precursors by the RNaseIII-like enzyme Dicer. In C. elegans, miRNAs disrupt translation of target mRNAs through imperfect base pairing to multiple sites within the 3′-UTR. Three research groups [ 1. Llave C. et al. Endogenous and silencing-associated small RNAs in plants. Plant Cell. 2002; 14: 1605-1619 Crossref PubMed Scopus (738) Google Scholar , 2. Reinhart B.J. et al. MicroRNAs in plants. Genes Dev. 2002; 16: 1616-1626 Crossref PubMed Scopus (1638) Google Scholar , 3. Rhoades, M.W. et al. (2002) Prediction of plant microRNA targets. Cell. Published online 31 July 2002. DOI: 10.1016/S0092867402008632 (http://www.cell.com) Google Scholar ] now show for the first time that similar small RNAs are also present in plants, probably using a PTGS-like mechanism to regulate plant development.