ArticleLiterature Review

Almeida, R and Allshire, RC. RNA silencing and genome regulation. Trends Cell Biol 15: 251-258

June 2005
Trends in Cell Biology 15(5):251-8

June 2005
15(5):251-8

DOI:10.1016/j.tcb.2005.03.006

Source
PubMed

Authors:

Ricardo Almeida

UCSF University of California, San Francisco

Robin Allshire

The University of Edinburgh

Closely related RNA silencing phenomena such as posttranscriptional and transcriptional gene silencing (PTGS and TGS), quelling and RNA interference (RNAi) represent different forms of a conserved ancestral process. The biological relevance of these RNA-directed mechanisms of silencing in gene regulation, genome defence and chromosomal structure is rapidly being unravelled. Here, we review the recent developments in the field of RNA silencing in relation to other epigenetic phenomena and discuss the significance of this process and its targets in the regulation of modern eukaryotic genomes.

Editorial focus: Understanding off-target effects as the key to successful RNAi therapy

Article

Full-text available

Dec 2019

With the first RNA interference (RNAi) drug (ONPATTRO (patisiran)) on the market, we witness the RNAi therapy field reaching a critical turning point, when further improvements in drug candidate design and delivery pipelines should enable fast delivery of novel life changing treatments to patients. Nevertheless, ignoring parallel development of RNAi dedicated in vitro pharmacological profiling aiming to identify undesirable off-target activity may slow down or halt progress in the RNAi field. Since academic research is currently fueling the RNAi development pipeline with new therapeutic options, the objective of this article is to briefly summarize the basics of RNAi therapy, as well as to discuss how to translate basic research into better understanding of related drug candidate safety profiles early in the process.

siRNADesign: A Graph Neural Network for siRNA Efficacy Prediction via Deep RNA Sequence Analysis

Preprint

Apr 2024

With the growing attention on siRNA silencing efficacy prediction, many methods have been proposed recently ranging from traditional data analysis methods to advanced machine learning models. However, previous works fail to explore complex but vital information, e.g., the RNA sequence interactions and related proteins. To alleviate this issue, we propose siRNADesign, a GNN model that innovatively analyzes both non-empirical and empirical-rules-based features of siRNA and mRNA sequences. This comprehensive approach allows siRNADesign to capture the nuanced dynamics of gene silencing effectively, achieving unprecedented state-of-the-art results across various datasets. Furthermore, we introduce a novel dataset-splitting methodology to mitigate the issues of data leakage and the shortcomings of traditional validation techniques in prior works. By considering siRNA and mRNA sequences as separate entities for dataset segmentation, this method guarantees a more accurate and unbiased evaluation of the model's performance. Through extensive evaluation on both widely used and external datasets, siRNADesign has demonstrated exceptional predictive accuracy and robustness under diverse experimental conditions. This work not only provides a robust foundation for the advancement of predictive models in gene silencing but also proposes a new dataset-splitting approach that aims to redefine the standards for future research, promoting more thorough and realistic assessment methodologies.

Therapeutic potential of plant-derived amiRNAs: challenges and strategies

Article

Full-text available

Jan 2024

RNAi-Based Therapy: Combating Shrimp Viral Diseases

Article

Full-text available

Oct 2023

Shrimp aquaculture has become a vital industry, meeting the growing global demand for seafood. Shrimp viral diseases have posed significant challenges to the aquaculture industry, causing major economic losses worldwide. Conventional treatment methods have proven to be ineffective in controlling these diseases. However, recent advances in RNA interference (RNAi) technology have opened new possibilities for combating shrimp viral diseases. This cutting-edge technology uses cellular machinery to silence specific viral genes, preventing viral replication and spread. Numerous studies have shown the effectiveness of RNAi-based therapies in various model organisms, paving the way for their use in shrimp health. By precisely targeting viral pathogens, RNAi has the potential to provide a sustainable and environmentally friendly solution to combat viral diseases in shrimp aquaculture. This review paper provides an overview of RNAi-based therapy and its potential as a game-changer for shrimp viral diseases. We discuss the principles of RNAi, its application in combating viral infections, and the current progress made in RNAi-based therapy for shrimp viral diseases. We also address the challenges and prospects of this innovative approach.

Biotechnological Interventions in Coldwater Aquaculture Health Management

Chapter

Sep 2023

Md. Idrish Raja Khan

Modern Approachesin Plant Plant Pathology Vol 2 230902 132527 (1)

Chapter

Full-text available

Sep 2023

Virupakshi Hiremata

miRNAs as biomarkers in human diseases

Article

Full-text available

Jan 2022

RNA interference (RNAi) is one of the primary machineries involved in the regulation of gene expression using small double-stranded RNA (dsRNA) in eukaryotic cells. MicroRNA (miRNA) is a class of small non-coding RNAs, regulating gene expression through canonical and non-canonical ways. Previous studies have shown that miRNA coding sequences make up 1% of the human genome and currently 1917 human miRNAs are displayed in the miRBase database. Expression levels of circulating miRNAs are related to various pathophysiological conditions such as cancer, infectious conditions, cardiovascular diseases, neurodegenerative diseases, and many more. Therefore, it is important to identify, detect and analyse miRNAs by using in silico and experimental analyses. In this review, after a brief description, we discuss the use of miRNAs for diagnosis and prognosis as biomarkers and biosensors in addition to miRNA-based therapies.

Analyses of gene copy number variation in diverse epigenetic regulatory gene families across plants: Increased copy numbers of BRUSHY1/TONSOKU/MGOUN3 (BRU1/TSK/MGO3) and SILENCING DEFECTIVE 3 (SDE3) in long-lived trees

Article

Sep 2022

Long-lived trees experience high risk of damage due to the various types of stresses over their lifespans. Epigenetic regulation is involved in gene regulation, genome integrity, and inhibition of exogenous genetic elements, which are functions important for long-term survival. To narrow down the candidate genes related to tree longevity among diverse epigenetic regulatory genes, it is necessary to identify epigenetic regulatory genes with increased copy number in long-lived tree species as compared to in short-lived annual and perennial herb species. In the present study, to find out the epigenetic regulatory genes with increased copy number in tree species as compared to in annual and perennial herb species, we conducted the systematic comparison of copy number variation in 121 gene families involved in various epigenetic regulatory pathways across 85 plant species with different lifespans using a genome database. Among these 121 gene families, the gene family encoding BRUSHY1/TONSOKU/MGOUN3 (BRU1/TSK/MGO3) and that encoding SILENCING DEFECTIVE 3 (SDE3) were found to exhibit significantly higher copy number of genes in tree species than in both perennial and annual herb species. BRU1/TSK/MGO3 is involved in chromatin modifications and plays an important role in the maintenance of meristems, genome integrity, and the inheritance of chromatin states. SDE3 is involved in RNA silencing and has an important role in antiviral defense through posttranscriptional gene silencing. The systematic comparison of copy number variation in diverse epigenetic regulatory gene families among plant species can find out epigenetic regulatory genes with increased copy number in long-lived tree species and enhance subsequent studies for understanding the relationship between epigenetic regulation and tree longevity.

Plant Viruses of Agricultural Importance: Current and Future Perspectives of Virus Disease Management Strategies

Article

Sep 2022
PHYTOPATHOLOGY

Plant viruses cause significant losses in agricultural crops worldwide, affecting the yield and quality of agricultural products. The emergence of novel viruses or variants through genetic evolution and spillover from reservoir host species, changes in agricultural practices, mixed infections with disease synergism, and impacts from global warming pose continuous challenges for the management of epidemics resulting from emerging plant virus diseases. This review describes some of the most devastating virus diseases plus select virus diseases with regional importance in agriculturally important crops that have caused significant yield losses. The lack of curative measures for plant virus infections prompts the use of risk-reducing measures for managing plant virus diseases. These measures have included exclusion, avoidance, and eradication techniques, along with vector management practices. The advent of Next-Generation Sequencing technologies has great potential for detecting unknown viruses in quarantine samples. The deployment of genetic resistance in crop plants is an effective and desirable method of managing virus diseases. Several dominant and recessive resistance genes have been used to manage virus diseases in crops. Recently, RNA-based technologies such as dsRNA- and siRNA-based RNA interference, microRNA, and CRISPR/Cas9 provide transgenic and non-transgenic approaches for developing virus-resistant crop plants. Importantly, the topical application of dsRNA, hairpin RNA, and artificial microRNA and trans-active siRNA molecules on plants has the potential to develop GMO-free virus disease management methods. However, the long-term efficacy and acceptance of these new technologies, especially transgenic methods, remain to be established

Parasitic plant small RNA analyses unveil parasite-specific signatures of microRNA retention, loss, and gain

Article

Full-text available

Jul 2022

Parasitism is a successful life strategy that has evolved independently in several families of vascular plants. The genera Cuscuta and Orobanche represent examples of the two profoundly different groups of parasites: one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs from Cuscuta campestris and Orobanche aegyptiaca. We showed that Cuscuta campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event. The exploratory analysis of the miRNA population (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic Orobanche aegyptiaca also revealed a loss of a number of miRNAs compared to photosynthetic species from the same order. In summary, our study shows partly similar evolutionary signatures in the RNA silencing machinery in both parasites. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants.

Novel mating-type-associated genes and gene fragments in the genomes of Mycosphaerellaceae and Teratosphaeriaceae fungi

Article

Apr 2022
MOL PHYLOGENET EVOL

The mating-type (MAT1) locus encodes transcription factors essential for the onset of the sexual cycle in ascomycete fungi. This locus has been characterised in only a few heterothallic, plant pathogenic Mycosphaerellaceae and Teratosphaeriaceae. We used available genome sequences for Mycosphaerellales species to investigate the presence of two unique mating-type-associated features. The accessory MAT1 genes, MAT1-1-10 (MATORF2) and MAT1-2-12 (MATORF1), typically occurred in both MAT idiomorphs of Mycosphaerellaceae species. In contrast, they were associated with only one idiomorph in Teratosphaeriaceae species. In Pseudocercospora, phylogenetic analyses showed that homologs present in different idiomorphs were paralogous and subject to different selective pressures, indicating that their evolution is linked to mating type. In almost half of the investigated Mycosphaerellales genomes, numerous short fragment sequences, almost identical to portions of the MAT1-1-1 and MAT1-2-1 genes, were present in multiple areas outside of the MAT1 locus. Aligned to the MAT1 genes, these sequences resembled an mRNA transcript. Fragment sequences were similar among species groups and occurred at the same genomic positions, implying that monophyletic groups have the same origins of these sequences. Although the functions of the MAT fragment sequences and accessory MAT1 genes remain unknown, both were expressed in the representative Mycosphaerellaceae and Teratosphaeriaceae species that were investigated.

MicroRNAs, Long Non-Coding RNAs, and Circular RNAs in the Redox Control of Cell Senescence

Article

Full-text available

Feb 2022

Cell senescence is critical in diverse aspects of organism life. It is involved in tissue development and homeostasis, as well as in tumor suppression. Consequently, it is tightly integrated with basic physiological processes during life. On the other hand, senescence is gradually being considered as a major contributor of organismal aging and age-related diseases. Increased oxidative stress is one of the main risk factors for cellular damages, and thus a driver of senescence. In fact, there is an intimate link between cell senescence and response to different types of cellular stress. Oxidative stress occurs when the production of reactive oxygen species/reactive nitrogen species (ROS/RNS) is not adequately detoxified by the antioxidant defense systems. Non-coding RNAs are endogenous transcripts that govern gene regulatory networks, thus impacting both physiological and pathological events. Among these molecules, microRNAs, long non-coding RNAs, and more recently circular RNAs are considered crucial mediators of almost all cellular processes, including those implicated in oxidative stress responses. Here, we will describe recent data on the link between ROS/RNS-induced senescence and the current knowledge on the role of non-coding RNAs in the senescence program.

Transposable Elements: Distribution, Polymorphism, and Climate Adaptation in Populus

Article

Full-text available

Feb 2022

Transposable elements (TEs) are a class of mobile genetic elements that make effects on shaping rapid phenotypic traits of adaptive significance. TE insertions are usually related to transcription changes of nearby genes, and thus may be subjected to purifying selection. Based on the available genome resources of Populus, we found that the composition of Helitron DNA family were highly variable and could directly influence the transcription of nearby gene expression, which are involving in stress-responsive, programmed cell death, and apoptosis pathway. Next, we indicated TEs are highly enriched in Populus trichocarpa compared with three other congeneric poplar species, especially located at untranslated regions (3′UTRs and 5′UTRs) and Helitron transposons, particularly 24-nt siRNA-targeted, are significantly associated with reduced gene expression. Additionally, we scanned a representative resequenced Populus tomentosa population, and identified 9,680 polymorphic TEs loci. More importantly, we identified a Helitron transposon located at the 3′UTR, which could reduce WRKY18 expression level. Our results highlight the importance of TE insertion events, which could regulate gene expression and drive adaptive phenotypic variation in Populus.

Biotechnological Approaches in Fish Health Management

Chapter

Jan 2021

With the advancement in aquaculture practices and the unresponsible approach towards the aquatic environment, the fisheries sector is suffering from severe disease outbreaks, resulting in significant losses globally. Consequently, disease management approaches targeted towards the pathogen are not enough and indeed not a sustainable option. Over the years, an array of biotechnological approaches has shown promising response to improve the health of cultured organisms and accomplish the greater production while safeguarding the aquatic environment. Such biotechnological interventions include pathogen-free best management practices by avoiding possible use of chemical remedial agents and applying biocontrol approaches against pathogens of diverse origin. This chapter highlights various biotechnological approaches to biocontrol disease outbreaks in aquaculture, like the adoption of vaccines or immunostimulants, probiotic, prebiotic, symbiotic, paraprobiotics, phage therapy, antimicrobial peptides, gene therapy, RNA interference, etc. The biotechnological tools outlined in this chapter could be of utmost importance to achieve the better health management of cultured aquatic organism and the long-term sustainable development goal of aquaculture.

Novel open reading frames in human accelerated regions and transposable elements reveal new leads to understand schizophrenia and bipolar disorder

Article

Full-text available

Mar 2022

Schizophrenia (SCZ) and bipolar disorder are debilitating neuropsychiatric disorders arising from a combination of environmental and genetic factors. Novel open reading frames (nORFs) are genomic loci that give rise to previously uncharacterized transcripts and protein products. In our previous work, we have shown that nORFs can be biologically regulated and that they may play a role in cancer and rare diseases. More importantly, we have shown that nORFs may emerge in accelerated regions of the genome giving rise to species-specific functions. We hypothesize that nORFs represent a potentially important group of biological factors that may contribute to SCZ and bipolar disorder pathophysiology. Human accelerated regions (HARs) are genomic features showing human-lineage-specific rapid evolution that may be involved in biological regulation and have additionally been found to associate with SCZ genes. Transposable elements (TEs) are another set of genomic features that have been shown to regulate gene expression. As with HARs, their relevance to SCZ has also been suggested. Here, nORFs are investigated in the context of HARs and TEs. This work shows that nORFs whose expression is disrupted in SCZ and bipolar disorder are in close proximity to HARs and TEs and that some of them are significantly associated with SCZ and bipolar disorder genomic hotspots. We also show that nORF encoded proteins can form structures and potentially constitute novel drug targets.

An Influence of Modification with Phosphoryl Guanidine Combined with a 2′-O-Methyl or 2′-Fluoro Group on the Small-Interfering-RNA Effect

Article

Full-text available

Sep 2021
INT J MOL SCI

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2′-O-methyl (2′-OMe) or PG/2′-fluoro (2′-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

Strategies to combat the problem of yam anthracnose disease: Status and prospects

Article

Full-text available

Jun 2021

Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devastating fungal disease of yam in West Africa, leading to 50%-90% of tuber yield losses in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. alatae affects all parts of the yam plant at all stages of development, including leaves, stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region and other yam-producing countries in the world. Traditional methods adopted by farmers to control the disease have not been very successful. Fungicides have also failed to provide long-lasting control. Although conventional breeding and genomics-assisted breeding have been used to develop some level of resistance to anthracnose in Dioscorea alata, the appearance of new and more virulent strains makes the development of improved varieties with broad-spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted researchers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus-resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose-resistant yam varieties for improved food security in West Africa. K E Y W O R D S anthracnose, CRISPR/Cas, Dioscorea spp., fungal diseases, genomics-assisted breeding, new breeding techniques, RNAi, yam

Strategies to combat the problem of yam anthracnose disease: Status and prospects

Article

Full-text available

Jul 2021
MOL PLANT PATHOL

Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devastating fungal disease of yam in West Africa, leading to 50%–90% of tuber yield losses in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. alatae affects all parts of the yam plant at all stages of development, including leaves, stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region and other yam‐producing countries in the world. Traditional methods adopted by farmers to control the disease have not been very successful. Fungicides have also failed to provide long‐lasting control. Although conventional breeding and genomics‐assisted breeding have been used to develop some level of resistance to anthracnose in Dioscorea alata, the appearance of new and more virulent strains makes the development of improved varieties with broad‐spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted researchers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus‐resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose‐resistant yam varieties for improved food security in West Africa. Resistance to yam anthracnose, the most devastating fungal disease of yam (Dioscorea spp.), can be produced by genomics‐assisted breeding, high‐throughput phenotyping, genetic engineering, and CRISPR/Cas9 genome editing.

Recent advances and emerging trends in antiviral defense networking in rice

Article

Full-text available

Mar 2021

Plants and viruses coexist in the natural ecosystem for extended periods of time, interacting with each other and even coevolving, maintaining a dynamic balance between plant disease resistance and virus pathogenicity. During virus–host interactions, plants often exhibit abnormal growth and development. However, plants do not passively withstand virus attacks but have evolved sophisticated and effective defense mechanisms to resist, limit, or undermine virus infections. It is widely believed that the initial stage of infection features the most intense interactions between the virus and the host and the greatest variety of activated signal transduction pathways. This review describes the most recent findings in rice antiviral research and discusses a variety of rice antiviral molecular mechanisms, including those based on R genes and recessive resistance, RNA silencing, phytohormone signaling, autophagy and WUS-mediated antiviral immunity. Finally, we discuss the challenges and future prospects of breeding rice for enhanced virus resistance.

Plant parasitic nematodes effectors and their crosstalk with defense response of host plants: A battle underground

Article

Nov 2020

Phytoparasitic nematodes are devastating pests that cause high yield losses in agriculture. A parasitic nematode injects effector molecules into root cells to manipulate the root cell processes. In response, plants activate their immune response against nematodes infection by recognizing the invasion through several different and complementary systems. Genomic techniques have identified putative nematode effector molecules that alter plant cell biology and induce giant cell formation. The nematode effector molecules are identified by plant cell surface recognition receptors, which cause pattern-triggered immunity. Plants recognize damage to a root cell caused by a nematode stylet with a set of molecules identified as a pattern of damage-associated gene expression. The plant immune response to parasitic nematodes includes defense genes activation, signal transduction, etc. In this review, we emphasize the role of nematode molecular effectors in parasitizing roots. We summarize the mechanisms of infection, signal transduction pathways implicated, and the elicited plant immune response.

A transmissible RNA pathway in honey bees

Preprint

Full-text available

Aug 2018

One of the characteristics of RNA interference (RNAi) is systemic spread of the silencing signal among cells and tissues throughout the organism. Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We show that ingested dsRNA spreads through the bee’s hemolymph associated with a protein complex. The systemic dsRNA is secreted with the jelly and delivered to larvae via ingestion. Furthermore, we demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Finally, RNA extracted from worker and royal jellies harbor differential naturally occurring RNA populations. Some of these RNAs corresponded to honey bee protein coding genes, transposable elements, non-coding RNA as well as bacteria, fungi and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Thus, our findings suggest a transmissible RNA pathway, playing a role in social immunity and epigenetic signaling between honey bees and potentially among other closely interacting organisms. SIGNIFICANCE Honey bees are eusocial insects, living in a colony that is often described as a superorganism. RNA mobility among cells of an organism has been documented in plants and animals. Here we show that RNA spreads further in honey bees, and is horizontally transferred between individuals and across generations. We found that honey bees share biologically active RNA through secretion and ingestion of worker and royal jellies. Such RNA initiates RNA interference, which is a known defense mechanism against viral infection. Furthermore, we characterized diverse RNA profiles of worker and royal jelly, including fragmented viral RNA. Our findings demonstrate a transmissible RNA pathway with potential roles in social immunity and epigenetic signaling among members of the hive.

Dengue Virus and the Relationship with MicroRNAs

Chapter

Full-text available

Jul 2020

Dengue is an acute febrile disease caused by a virus of the genus Flavivirus, family Flaviviridae, endemic in tropical regions of the globe. The agent is a virus with single-stranded RNA, classified into four distinct dengue virus (DENV) serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The host’s innate and adaptive immune responses play an essential role in determining the natural history of viral infections, especially in dengue. In this context, it has observed in recent years that the presence of RNA interference (RNAi) in viral infection processes is increasing, as well as immune defense. The context microRNAs (miRNAs) go for stood out, as their presence during viral infection, both in the replication of the virus and in the defense against these infections, becomes increasingly noticeable, therefore, making it increasingly necessary to better understand the role of these small RNAs within viral infection by DENV and what their consequences are in aggravating the consequences of patients affected by this disease.

Repeat induces not only gene silencing, but also gene activation in mammalian cells

Article

Full-text available

Jun 2020
PLOS ONE

Repeat-induced gene silencing (RIGS) establishes the centromere structure, prevents the spread of transposons and silences transgenes, thereby limiting recombinant protein production. We previously isolated a sequence (B-3-31) that alleviates RIGS from the human genome. Here, we developed an assay system for evaluating the influence of repeat sequences on gene expression, based on in vitro ligation followed by our original gene amplification technology in animal cells. Using this assay, we found that the repeat of B-3-31, three core sequences of replication initiation regions (G5, C12, and D8) and two matrix attachment regions (AR1 and 32–3), activated the co-amplified plasmid-encoded d2EGFP gene in both human and hamster cell lines. This upregulation effect persisted for up to 82 days, which was confirmed to be repeat-induced, and was thus designated as a repeat-induced gene activation (RIGA). In clear contrast, the repeat of three bacterial sequences (lambda-phage, Amp, and ColE1) and three human retroposon sequences (Alu, 5’-untranslated region, and ORF1 of a long interspersed nuclear element) suppressed gene expression, thus reflecting RIGS. RIGS was CpG-independent. We suggest that RIGA might be associated with replication initiation. The discovery of RIGS and RIGA has implications for the repeat in mammalian genome, as well as practical value in recombinant production.

Epigenetic changes and their relationship to somaclonal variation: a need to monitor the micropropagation of plantation crops

Article

Apr 2020

Chromatin modulation plays important roles in gene expression regulation and genome activities. In plants, epigenetic changes, including variations in histone modification and DNA methylation, are linked to alterations in gene expression. Despite the significance and potential of in vitro cell and tissue culture systems in fundamental research and marketable applications, these systems threaten the genetic and epigenetic networks of intact plant organs and tissues. Cell and tissue culture applications can lead to DNA variations, methylation alterations, transposon activation, and finally, somaclonal variations. In this review, we discuss the status of the current understanding of epigenomic changes that occur under in vitro conditions in plantation crops, including coconut, oil palm, rubber, cotton, coffee and tea. It is hoped that comprehensive knowledge of the molecular basis of these epigenomic variations will help researchers develop strategies to enhance the totipotent and embryogenic capabilities of tissue culture systems for plantation crops.

Pulmonary Administration: Strengthening the Value of Therapeutic Proximity

Article

Full-text available

Feb 2020

In recent years inhaled systems have shown momentum as patient-personalized therapies emerge. A significant improvement in terms of therapeutic efficacy and/or reduction adverse systemic effects is anticipated from their use owing these systems regional accumulation. Nevertheless, whatever safety and efficacy evidence required for inhaled formulations regulatory approval, it still poses an additional hurdle to gaining market access. In contrast with the formal intravenous medicines approval, the narrower adoption of pulmonary administration might rely on discrepancies in pre-clinical and clinical data provided by the marketing authorization holder to the regulatory authorities. Evidences of a diverse and inconsistent regulatory framework led to concerns over toxicity issues and respiratory safety. However, an overall trend to support general concepts of good practices exists. Current regulatory guidelines1 that supports PK/PD (pharmacokinetics/pharmacodynamic) assessment seeks attention threatening those inhaled formulations set to be approved in the coming years. A more complex scenario arises from the attempt of implementing nanomedicines for pulmonary administration. Cutting-edge image techniques could play a key role in supporting diverse stages of clinical development facilitating this pharmaceutics take off and speed to patients. The ongoing challenge in adapting conventional regulatory frameworks has proven to be tremendously difficult in an environment where market entry relies on multiple collections of evidence. This paper intention is to remind us that an acceptable pre-clinical toxicological program could emerge from, but not only, an accurate and robust data imaging collection. It is our conviction that if implemented, inhaled nanomedicines might have impact in multiple severe conditions, such as lung cancer, by fulfilling the opportunity for developing tailored treatments while solving dose-related toxicity issues; the most limiting threat in conventional lung cancer clinical management.

Recent Advances in the Treatment of Cerebellar Disorders

Article

Full-text available

Dec 2019
BSRCCS

Various etiopathologies affect the cerebellum, resulting in the development of cerebellar ataxias (CAs), a heterogeneous group of disorders characterized clinically by movement incoordination, affective dysregulation, and cognitive dysmetria. Recent progress in clinical and basic research has opened the door of the ‘‘era of therapy” of CAs. The therapeutic rationale of cerebellar diseases takes into account the capacity of the cerebellum to compensate for pathology and restoration, which is collectively termed cerebellar reserve. In general, treatments of CAs are classified into two categories: cause-cure treatments, aimed at arresting disease progression, and neuromodulation therapies, aimed at potentiating cerebellar reserve. Both forms of therapies should be introduced as soon as possible, at a time where cerebellar reserve is still preserved. Clinical studies have established evidence-based cause-cure treatments for metabolic and immune-mediated CAs. Elaborate protocols of rehabilitation and non-invasive cerebellar stimulation facilitate cerebellar reserve, leading to recovery in the case of controllable pathologies (metabolic and immune-mediated CAs) and delay of disease progression in the case of uncontrollable pathologies (degenerative CAs). Furthermore, recent advances in molecular biology have encouraged the development of new forms of therapies: the molecular targeting therapy, which manipulates impaired RNA or proteins, and the neurotransplantation therapy, which delays cell degeneration and facilitates compensatory functions. The present review focuses on the therapeutic rationales of these recently developed therapeutic modalities, highlighting the underlying pathogenesis.

Contrôle de la différenciation sexuelle de la levure Schizosaccharomyces pombe par un ARN non-codant et la protéine de liaison à l’ARN Mmi1

Thesis

Nov 2017

Mathieu Dangin

Au cours des cinq dernières années l’existence d’un contrôle de la transcription par les ARN non-codants longs (lncRNAs) a été décrite dans une large variété d’eucaryotes. Cependant, les mécanismes par lesquels les lncRNAs régulent la transcription restent en grande partie méconnus. Les premiers travaux effectués dans le cadre de cette thèse ont participé à la caractérisation du mécanisme mis en jeu par un lncRNA, nommé nam1, dans le contrôle de l’entrée en différenciation sexuelle chez la levure Schizosaccharomyces pombe. Il a ainsi été montré qu’au cours de sa synthèse le lncRNA nam1 est ciblé par la protéine de liaison à l’ARN Mmi1 et une machinerie de surveillance des ARN qui comprend l’exosome, un complexe de dégradation des ARN conservé au cours de l’évolution. La fixation de Mmi1 au lncRNA nam1 contrôle la terminaison de la transcription de nam1 et empêche ainsi la transcription de se poursuivre et d’interférer alors avec la transcription du gène situé en aval (codant pour une MAP kinase essentielle à l’entrée en différenciation). Les travaux suivant montrent l’implication dans ce mécanisme de la protéine Cti1, un des co-facteurs connus de l’exosome. Fait marquant, ces travaux rapportent aussi l’existence d’un mode de production inédit pour un lncRNA. En effet, ils révèlent que la transcription non-interrompue d’un gène codant conduirait à la production d’un ARN bi-cistronique. La maturation co-transcriptionnelle de cet ARN bi-cistronique produirait, d’un côté, un ARN messager et, de l’autre, le lncRNA nam1. Enfin, ils ont permit la caractérisation initiale d’un nouveau composant de la machinerie de surveillance des ARN recrutée sur nam1 par Mmi1. Ainsi, dans leur ensemble, ces travaux contribuent à une meilleure connaissance des mécanismes pouvant être mis en jeu par un lncRNA et agissant en cis pour réguler l’expression génique et, à travers elle, des processus cellulaires majeurs, tel que la différenciation cellulaire. De plus, ils décrivent un nouveau mécanisme de biogénèse d’un lncRNA.

Digitizable therapeutics for decentralized mitigation of global pandemics

Article

Full-text available

Oct 2019

When confronted with a globally spreading epidemic, we seek efficient strategies for drug dissemination, creating a competition between supply and demand at a global scale. Propagating along similar networks, e.g., air-transportation, the spreading dynamics of the supply vs. the demand are, however, fundamentally different, with the pathogens driven by contagion dynamics, and the drugs by commodity flow. We show that these different dynamics lead to intrinsically distinct spreading patterns: while viruses spread homogeneously across all destinations, creating a concurrent global demand, commodity flow unavoidably leads to a highly uneven spread, in which selected nodes are rapidly supplied, while the majority remains deprived. Consequently, even under ideal conditions of extreme production and shipping capacities, due to the inherent heterogeneity of network-based commodity flow, efficient mitigation becomes practically unattainable, as homogeneous demand is met by highly heterogeneous supply. Therefore, we propose here a decentralized mitigation strategy, based on local production and dissemination of therapeutics, that, in effect, bypasses the existing distribution networks. Such decentralization is enabled thanks to the recent development of digitizable therapeutics, based on, e.g., short DNA sequences or printable chemical compounds, that can be distributed as digital sequence files and synthesized on location via DNA/3D printing technology. We test our decentralized mitigation under extremely challenging conditions, such as suppressed local production rates or low therapeutic efficacy, and find that thanks to its homogeneous nature, it consistently outperforms the centralized alternative, saving many more lives with significantly less resources.

siRNA-Finder (si-Fi) Software for RNAi-Target Design and Off-Target Prediction

Article

Full-text available

Aug 2019

RNA interference (RNAi) is a technique used for transgene-mediated gene silencing based on the mechanism of posttranscriptional gene silencing (PTGS). PTGS is an ubiquitous basic biological phenomenon involved in the regulation of transcript abundance and plants’ immune response to viruses. PTGS also mediates genomic stability by silencing of retroelements. RNAi has become an important research tool for studying gene function by strong and selective suppression of target genes. Here, we present si-Fi, a software tool for design optimization of RNAi constructs necessary for specific target gene knockdown. It offers efficiency prediction of RNAi sequences and off-target search, required for the practical application of RNAi. si-Fi is an open-source (CC BY-SA license) desktop software that works in Microsoft Windows environment and can use custom sequence databases in standard FASTA format.

miRNAs in depression vulnerability and resilience: novel targets for preventive strategies

Article

Full-text available

Sep 2019
J NEURAL TRANSM

The exposure to stressful experiences during the prenatal period and through the first years of life is known to affect the brain developmental trajectories, leading to an enhanced vulnerability for the development of several psychiatric disorders later in life. However, not all the subjects exposed to the same stressful experience develop a pathologic condition, as some of them, activating coping strategies, become more resilient. The disclosure of mechanisms associated with stress vulnerability or resilience may allow the identification of novel biological processes and potential molecules that, if properly targeted, may prevent susceptibility or potentiate resilience. Over the last years, miRNAs have been proposed as one of the epigenetic mechanisms mediating the long-lasting effects of stress. Accordingly, they are associated with the development of stress vulnerability or resilience-related strategies. Moreover, miRNAs have been proposed as possible biomarkers able to identify subjects at high risk to develop depression and to predict the response to pharmacological treatments. In this review, we aimed to provide an overview of findings from studies in rodents and humans focused on the involvement of miRNAs in the mechanisms of stress response with the final goal to identify distinct sets of miRNAs involved in stress vulnerability or resilience. In addition, we reviewed studies on alterations of miRNAs in the context of depression, showing data on the involvement of miRNAs in the pathogenesis of the disease and in the efficacy of pharmacological treatments, discussing the potential utility of miRNAs as peripheral biomarkers able to predict the treatment response.

Recent Advances in DNA Methylation and Their Potential Breeding Applications in Plants

Article

Jun 2022

Citation: Shaikh, A.A.; Chachar, S.; Chachar, M.; Ahmed, N.; Guan, C.; Zhang, P. Recent Advances in DNA Methylation and Their Potential Breeding Applications in Plants. Horticulturae 2022, 8, 562. https://doi.org/10.3390/ horticulturae8070562 Academic Editor: Jose V. Die

Controlled Delivery of Target-Specific MicroRNA Analogs as a Key to RNAi Therapeutics in Cancer

Chapter

Jan 2024

Although the tumor microenvironment is well known to be important for cancer progression, understanding how the complex molecular networks contribute to cancers, particularly with all the different types of cancers, remains a limitation that often prevents the successful translation of research results in effective clinical applications. One approach has been the use of RNAi therapeutics. The exciting potential of these therapies is currently hampered by the high risk of off-target effects due to the imperfect complementarity used by these noncoding RNAs (ncRNAs) to recognize their mRNA targets. While this suggests that the miRNA analog specificity is one problem, another major obstacle is the problem of developing effective and targeted delivery systems specifically for the tumor that avoids serious adverse complications. These roadblocks indicate the need for the development of novel miRNA delivery systems and target-specific miRNA analogs before successful miRNA anticancer therapies become available. In this chapter, we discuss the current limitations of various types of miRNA analogs and the variety of delivery systems that are currently being developed.

Pulmonary Delivery of Nucleic Acids

Chapter

Jan 2024

Pulmonary diseases affect millions of people worldwide and elicit symptoms that may evolve into severe conditions. Despite currently recommended treatments that can successfully control mild to moderate symptoms, targeted therapy has the potential to improve serious lung conditions over long time spans. Nucleic acid therapy has shown remarkable efficacy outcomes against pulmonary diseases through its mechanism of modulating intracellular gene expression. To improve the stability of nucleic acid molecules under physiological conditions and achieve targeted delivery, nucleic acids engineered in drug-delivery systems and administered by inhalation have been investigated in recent years. In this chapter, we will review the recent advances in the inhalation of nucleic acid-based therapies for lung disorders. The manufacturing of nucleic acid-based nanosystems for inhalation, as well as their potential to overcome physiological barriers, will be discussed. Ultimately, we will report on studies on innovative inhaled formulations that achieved promising results in animals or in clinical trials and summarize the promising strategies for pulmonary diseases.

Plant MicroRNAs and Stress Response

Book

Full-text available

May 2023

MicroRNAs (miRNAs) are small (20–24 nt), single stranded, regulatory RNA molecules or gene regulators of critical transcriptional or post-transcriptional gene regulation in plants in sequence-specific order that respond to numerous abiotic stresses and animals, non-coding, highly evolutionarily conserved and widely distributed throughout the plant kingdom. MiRNAs are master regulators of plant growth and development, development attenuation under various environmental stresses by stress-responsive miRNAs and plant stress responses and tolerance. Drought, salinity, heat, cold, UV radiation, heavy metal, pathogens, pests and other microbial infections affect survival, growth, development, quality, yield, and production of plants. Stress induced miRNAs down regulate their target miRNAs. This down regulation leads to the accumulation and function of positive regulators, highlighting their roles in stress responses and tolerance. Plant miRNA mediated modifications include overexpression or repression of stress-responsive miRNAs and/or their target complementary or partially complementary gene products, miRNA-resistant target genes, target-mimics and artificial miRNAs. Thus, miRNAs may serve as "genomic gold mines", novel, potent and potential targets in plant genetic manipulations and miRNA-based biotechnology will aid plant improvement and crop-plant tolerance to different environmental stresses. This book reviews our recent understanding of plant microRNAs, biogenesis and functions, computational tools and bioinformatics, regulation of plant growth and development, expression studies, and the role of plant miRNAs in various biotic and abiotic stress-response regulation in plants.

RNA Interference–Based Gene Therapy Strategies for the Treatment of HIV

Chapter

Jan 2010

Integrated RNA-seq and RNAi Analysis of the Roles of the Hsp70 and SP Genes in Red-Shell Meretrix meretrix Tolerance to the Pathogen Vibrio parahaemolyticus

Article

Full-text available

Aug 2022
MAR BIOTECHNOL

The “Wanlihong” Meretrix meretrix (WLH-M) clam is a new variety of this species that has a red shell and stronger Vibrio tolerance than ordinary M. meretrix (ORI-M). To investigate the molecular mechanisms responsible for the WLH-M strain’s tolerance to Vibrio, we challenged clams with Vibrio parahaemolyticus and then assessed physiological indexes and conducted transcriptome analysis and RNA interference experiments. The mortality, tissue bacterial load, and hemocyte reactive oxygen species level of ORI-M were significantly higher than those of WLH-M, whereas the content and activity of lysozyme were significantly lower. Gene Ontology functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that immune and metabolic pathways were enriched in Vibrio-challenged clams. The expressions of the heat shock protein 70 (Hsp70) and serine protease (SP) genes, which are involved in antibacterial immunity, were significantly upregulated in WLH-M but not in ORI-M, while the expression of the kynurenine 3-monooxygenase gene, a proinflammatory factor, was significantly downregulated in WLH-M. RNA interference experiments confirmed that Hsp70 and SP downregulation could result in increased mortality of WLH-M. Therefore, we speculate that Hsp70 and SP may be involved in the antibacterial immunity of WLH-M in vivo. Our data provided a valuable resource for further studies of the antibacterial mechanism of WLH-M and provided a foundation for the breeding of pathogen-resistant strains.

Recent Advances in DNA Methylation and Their Potential Breeding Applications in Plants

Article

Full-text available

Jun 2022

Traditional plant breeding encompasses repetitive crossing and selection based on morphological traits, while phenotypic selection has been complemented by molecular methods in recent decades. Genome editing with techniques like the CRISPR-Cas9 system is still a novel approach that is being used to make direct modifications to nucleotide sequences of crops. In addition to these genetic alterations, an improved understanding of epigenetic variations such as DNA methylation on the phenotype of plants has led to increased opportunities to accelerate crop improvement. DNA methylation is the most widely studied epigenetic mark in plants and other eukaryotes. These epigenetic marks are highly conserved and involved in altering the activities and functions of developmental signals by catalyzing changes in the chromatin structure through methylation and demethylation. Cytosine methylation (5mC) is the most prevalent modification found in DNA. However, recent identification of N6-methyladenosine (6mA) in plants starts to reveal their critical role in plant development. Epigenetic modifications are actively involved in creating the phenotype by controlling essential biological mechanisms. Epigenetic modifications could be heritable and metastable causing variation in epigenetic status between or within species. However, both genetic and heritable epigenetic variation has the potential to drive natural variation. Hence, epigenome editing might help overcome some of the shortcomings of genome editing (such as gene knockout), which can have significant off-target effects and only enables the loss of a gene’s function. In this review, we have discussed the mechanism underlying DNA methylation and demethylation in plants. Methyltransferases and demethylases are involved in catalyzing specific types of modification. We also discuss the potential role of DNA modifications in crop improvement for meeting the requirements of sustainable and green agriculture.

Current Research and Innovations in Plant Pathology

Book

Full-text available

Jan 2022

Current Research and Innovations in Plant Pathology

Chapter

Full-text available

Jan 2022

Raheeba Tun Nisa

Hormone signalling in Plant defense

Research Trends in Agriculture Science,

Book

Oct 2021

Study of the effects of nanoparticles on the immune system

Thesis

Full-text available

Dec 2020

Arindam K Dey

The unique characteristics of nanomaterials (NMs) are valuable in many industrial and biomedical applications. However, NMs might also give rise to unforeseen toxicity that could adversely affect the biological system specially the immune system. In the immune system as phagocytes can engulf foreign materials therefore they are appropriate for immunotoxicity screening because these cells participate in unspecific and specific immune responses.In this study, we investigated the impact of different NMs, such as gold nanoparticles (AuNPs), neutral lipid nanoparticles (NLCs), cationic lipid nanoparticles (CLCs), and amphiphilic dendrimers (ADs) on primary macrophages (BMDMs) and dendritic cells (BMDCs) by two approaches. In the first approach, we investigated the direct effect of exposure to NMs. In the second approach, we studied the indirect effect by activating the NMs exposed cells with different activators (LPS and IL-4).The study of direct effect of NMs shows that none of the tested NMs altered the phagocytosis capacity. All of the NMs had none or little direct effect on BMDCs activation. In case of BMDMs, AuNPs, CLCs, and ADs significantly decreased cell activation while NLCs did not. The study of cytokine production based on IL-6 and TNF- production, NO production, and cellular metabolism based on the investigation of glycolysis and mitochondrial metabolism also indicated no or little direct effect on BMDCs and BMDMs. However, we have noticed a significant increase in MCP-1 production by CLCs and ADs exposed cells while AuNPs and NLCs did not modulate MCP-1 production. Also, we noted that CLCs and NLCs at high concentrations could slightly increase ROS production while AuNPs and ADs did not modulate ROS production by BMDMs and BMDCs.Investigation of indirect effect of NMs revealed, when NMs exposed cells were challenged with LPS, we have recorded NMs specific modulation in the response of BMDCs based on cell activation, cytokine and chemokine secretion, NO, and ROS production. All NMs showed none or little modification of cellular metabolism of BMDCs. On the other hand, we have recorded a significant alteration in NMs exposed BMDMs upon LPS challenge in response to cell activation, cytokine and chemokine secretion, NO production, and cellular metabolism. However, ROS production remains unaltered in the case of all the NMs exposed BMDMs.The study of in vitro antigen presentation, revealed that exposure to different NMs altered T cell-specific cytokines reflecting alteration in CD4 + T lymphocytes polarization.Overall, these results demonstrate that NMs directly do not modify phagocytic activity, cytokine and chemokine production, NO and ROS production, cellular metabolism, but when instigated with an activation signal like LPS or IL-4, NMs exposed cells respond differently.The outcome of this study enriches the knowledge regarding NMs specific immunotoxicity, which may facilitate the design of nanomaterials that retain their useful properties, but display reduced toxicity (i.e., safety-by-design). This research will also improve our understanding of how different NMs can modulate the immune system, which might help suggest new therapy acting on the immune system such as antiinflammatory drugs or vaccines.

Experimental MicroRNA Targeting Validation

Chapter

Aug 2021

microRNAs (miRNAs) have recently been recognized as a new dimension of posttranscriptional regulation. It is well defined that most human protein-coding genes are regulated by one or more miRNAs. Therefore, it is crucial to identify genes targeted by the miRNAs to better understand their functions. Although bioinformatics tools have the ability to identify target candidates it is still essential to identify physiological targets by experimental approaches. Currently, the majority of miRNA-target experimental validation approaches assess the changes in target expression in mRNA or protein level upon miRNA upregulation or downregulation. Additionally, finding out direct physical interactions between miRNAs and their targets is also among the experimental techniques. In this chapter we reviewed the existing experimental techniques for miRNA target identification by considering their advantages and potential drawbacks.

Hemocyte siRNA uptake is increased by 5′ cholesterol-TEG addition in Biomphalaria glabrata , snail vector of schistosome

Article

Full-text available

Feb 2021

Biomphalaria glabrata is one of the snail intermediate hosts of Schistosoma mansoni , the causative agent of intestinal schistosomiasis disease. Numerous molecular studies using comparative approaches between susceptible and resistant snails to S. mansoni infection have helped identify numerous snail key candidates supporting such susceptible/resistant status. The functional approach using RNA interference (RNAi) remains crucial to validate the function of such candidates. CRISPR-Cas systems are still under development in many laboratories, and RNA interference remains the best tool to study B. glabrata snail genetics. Herein, we describe the use of modified small interfering RNA (siRNA) molecules to enhance cell delivery, especially into hemocytes, the snail immune cells. Modification of siRNA with 5′ Cholesteryl TriEthylene Glycol (Chol-TEG) promotes cellular uptake by hemocytes, nearly eightfold over that of unmodified siRNA. FACS analysis reveals that more than 50% of hemocytes have internalized Chol-TEG siRNA conjugated to Cy3 fluorophores, 2 hours only after in vivo injection into snails. Chol-TEG siRNA targeting BgTEP1 (ThioEster-containing Protein), a parasite binding protein, reduced BgTEP1 transcript expression by 70–80% compared to control. The level of BgTEP1 protein secreted in the hemolymph was also decreased. However, despite the BgTEP1 knock-down at both RNA and protein levels, snail compatibility with its sympatric parasite is not affected suggesting functional redundancy among the BgTEP genes family in snail-schistosoma interaction.

Drosophila melanogaster in Glycobiology: Their Mutants Are Excellent Models for Human Diseases

Chapter

Jan 2020

The fruit fly Drosophila melanogaster is a holometabolous insect that lives in all warm countries. Drosophila serves as an excellent model organism for genetic studies as it has a short life cycle, produces a large number of offspring, has polytene chromosomes and can be maintained at a low cost. As such, Drosophila has become one of the most important model organisms in the fields of classical and molecular genetics as well as developmental biology. Drosophila have also been harnessed in the field of glycobiology to clarify the novel functions of glycans. Indeed, genome-wide screening and functional analyses of glycans have been performed in the context of the whole fly body, as well as the wings, eyes, neuromuscular junctions, and immune organs. Detailed studies using Drosophila mutants of glycosyltransferases, nucleotide sugar transporters, and glycosidases have revealed novel functions of N-linked glycans, glycosaminoglycans, glycolipids, and O-linked glycans including mucin type O-glycan, O-Fuc, O-Man, and O-GlcNAc. Because many of these functions are common between Drosophila and humans, these mutants serve as excellent models for human disease. In this review, We provide a brief introduction to Drosophila melanogaster and then provide an overview of Drosophila glycobiology.

Current Research and Innovations in Plant Pathology

Chapter

Full-text available

Dec 2020

The Effects of Single Nucleotide Polymorphisms in Cancer RNAi Therapies

Article

Full-text available

Oct 2020

Simple Summary Despite the recent progress in RNAi delivery of siRNA-based therapeutics for cancer therapy, the presence of single nucleotide polymorphisms (SNPs) in the general population could dramatically reduce the effectiveness of RNAi therapy. Their ubiquitous presence can also lead to unpredictable and adverse side effects. Because both SNPs and somatic mosaicisms have also been implicated in a number of human diseases including cancer, however, these specific changes offer the ability to selectively and efficiently target cancer cells. Here, we discuss how SNPs influence the development and success of novel anticancer RNAi therapies. Abstract Tremendous progress in RNAi delivery methods and design has allowed for the effective development of siRNA-based therapeutics that are currently under clinical investigation for various cancer treatments. This approach has the potential to revolutionize cancer therapy by providing the ability to specifically downregulate or upregulate the mRNA of any protein of interest. This exquisite specificity, unfortunately, also has a downside. Genetic variations in the human population are common because of the presence of single nucleotide polymorphisms (SNPs). SNPs lead to synonymous and non-synonymous changes and they occur once in every 300 base pairs in both coding and non-coding regions in the human genome. Much less common are the somatic mosaicism variations associated with genetically distinct populations of cells within an individual that is derived from postzygotic mutations. These heterogeneities in the population can affect the RNAi’s efficacy or more problematically, which can lead to unpredictable and sometimes adverse side effects. From a more positive viewpoint, both SNPs and somatic mosaicisms have also been implicated in human diseases, including cancer, and these specific changes could offer the ability to effectively and, more importantly, selectively target the cancer cells. In this review, we discuss how SNPs in the human population can influence the development and success of novel anticancer RNAi therapies and the importance of why SNPs should be carefully considered.

Postharvest Losses: A Global Threatening Key Hole for Food Safety and Security

Chapter

Full-text available

Jul 2020

In globally, the population was increasing a rapid manner with 3.5%/ten years. In India, 1947-2020, regarding population from 0.3-1.25 billion. But our production is increased only during the period of green revolution after attained a stagnant position. In last 25 years, production was stabled conditions. During every year, agricultural durables and perishables viz., food grains (14 million tons/16 $ 16,000 million); fruits and vegetables (40 million tons/$ 13,000 billion) was attempted lagging phase in production due to the postharvest losses by improper management, insufficient infrastructures, pest and diseases, lacking knowledge for implementation of new technologies in postharvest handling and processing. In this study described about, the postharvest loss’s development state, casual factors and mechanisms and management. Naturally, highly perishable agro-products like fruits and vegetables were attained the severe losses by pathogens and abiotic factors. Initially, these are managed through practices viz., correct time of harvesting, protect the products in well sanitized storage units, physical, chemical and biological treatments were noted that better management for avoiding the postharvest losses during pre to postharvest stages of crops.

Current Research and Innovations in Plant Pathology

Chapter

Jun 2020

Viral disease of tomato crops (Solanum lycopesicum L.): an overview

Article

May 2020

Global production of tomato has been hampered by the increased incidences of tomato viral disease. The high genetic heterogeneity of tomato plant viruses, because of their high mutation rates, has lead to ineffective control strategies and the fast spread of the viruses. Viruses utilize the resources in host plants for their replication. Therefore, identification and removal of the non-redundant proteins in the tomato plant based on the biological properties of the virus combined with an RNAi strategy may be a future control strategy. In this review, fourteen tomato viral diseases and their causal agents are reviewed and the control strategies for tomato viral diseases are discussed.

Research Journal of Agricultural Sciences RNA Interference Technology: A Novel Approach for Fruit and Vegetable Crop Improvement

Article

Full-text available

Oct 2019

A model for RNA-mediated gene silencing in higher plants.

Article

Full-text available

May 1998

Methylation-Mediated Transcriptional Silencing in Euchromatin by Methyl-CpG Binding Protein MBD1 Isoforms

Article

Full-text available

Sep 1999

DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16 , VHL , and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

RNA interference in Trypanosoma brucei: Cloning of small interfering RNAs provides evidence for retroposon-derived 24–26-nucleotide RNAs

Article

Full-text available

Nov 2001
RNA

Appolinaire Djikeng

In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21–25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma brucei by expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 × g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20–30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24–26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24–26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24–26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.

Smardon A, Spoerke JM, Stacey SC, Klein ME, Mackin N, Maine EM. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans. Curr Biol 10: 169-178

Article

Full-text available

Mar 2000
CURR BIOL

Background: Cell-fate determination requires that cells choose between alternative developmental pathways. For example, germ cells in the nematode worm Caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. Germ-line mitosis depends on a somatic signal that is mediated by a Notch-type signaling pathway. The ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for oogenesis. Here, we provide more insight into the role of ego-1 in germ-line development.Results: We have determined the ego-1 gene structure and the molecular basis of ego-1 alleles. Putative ego-1 null mutants had multiple, previously unreported defects in germ-line development. The ego-1 transcript was found predominantly in the germ line. The predicted EGO-1 protein was found to be related to the tomato RNA-directed RNA polymerase (RdRP) and to Neurospora crassa QDE-1, two proteins implicated in post-transcriptional gene silencing (PTGS). For a number of germ-line-expressed genes, ego-1 mutants were resistant to a form of PTGS called RNA interference.Conclusions: The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery.

Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression

Article

Full-text available

May 1990

To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits.

RNA-directed de novo methylation of genomic sequences in plants

Article

Full-text available

Mar 1994
CELL

One monomeric and three oligomeric potato spindle tuber viroid (PSTVd) cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation. Southern analysis of the integrates revealed that only their PSTVd-specific sequences become fully methylated, whereas the flanking T-DNA and the genomic plant DNA remain unaltered. Viroid cDNA methylation could only be observed after autonomous viroid RNA-RNA replication had taken place in these plants. These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA.

Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation

Article

Full-text available

Jul 1996

The molecular mechanisms involved in transgene-induced gene silencing ('quelling') in Neurospora crassa were investigated using the carotenoid biosynthetic gene albino-1 (al-1) as a visual marker. Deletion derivatives of the al-1 gene showed that a transgene must contain at least approximately 132 bp of sequences homologous to the transcribed region of the native gene in order to induce quelling. Transgenes containing only al-1 promoter sequences do not cause quelling. Specific sequences are not required for gene silencing, as different regions of the al-1 gene produced quelling. A mutant defective in cytosine methylation (dim-2) exhibited normal frequencies and degrees of silencing, indicating that cytosine methylation is not responsible for quelling, despite the fact that methylation of transgene sequences frequently is correlated with silencing. Silencing was shown to be a dominant trait, operative in heterokaryotic strains containing a mixture of transgenic and non-transgenic nuclei. This result indicates that a diffusable, trans-acting molecule is involved in quelling. A transgene-derived, sense RNA was detected in quelled strains and was found to be absent in their revertants. These data are consistent with a model in which an RNA-DNA or RNA-RNA interaction is involved in transgene-induced gene silencing in Neurospora.

RNA-Mediated RNA Degradation and Chalcone Synthase A Silencing in Petunia

Article

Full-text available

Apr 1997
CELL

Transgenic Petunia plants with a chsA coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional chsA RNA degradation. In these plants, shorter poly(A)+ and poly(A)- chsA RNAs are found, and a 3' end-specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the chsA 3' coding region and its 3' untranslated region. Equivalent chsA RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA-RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.

Transposable elements as sources of variation in animals

Article

Full-text available

Aug 1997

A tremendous wealth of data is accumulating on the variety and distribution of transposable elements (TEs) in natural populations. There is little doubt that TEs provide new genetic variation on a scale, and with a degree of sophistication, previously unimagined. There are many examples of mutations and other types of genetic variation associated with the activity of mobile elements. Mutant phenotypes range from subtle changes in tissue specificity to dramatic alterations in the development and organization of tissues and organs. Such changes can occur because of insertions in coding regions, but the more sophisticated TE-mediated changes are more often the result of insertions into 5' flanking regions and introns. Here, TE-induced variation is viewed from three evolutionary perspectives that are not mutually exclusive. First, variation resulting from the intrinsic parasitic nature of TE activity is examined. Second, we describe possible coadaptations between elements and their hosts that appear to have evolved because of selection to reduce the deleterious effects of new insertions on host fitness. Finally, some possible cases are explored in which the capacity of TEs to generate variation has been exploited by their hosts. The number of well documented cases in which element sequences appear to confer useful traits on the host, although small, is growing rapidly.

Systemic acquired silencing: Transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions

Article

Full-text available

Sep 1997

Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and transgenes and of post-transcriptional silencing of a uidA transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding transgene. Transmission is unidirectional from stock to scion, transgene specific, locus independent and requires the presence of a transcriptionally active transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.

Wassenegger, M. & Pelissier, T. A model for RNA-mediated gene silencing in higher plants. Plant Mol. Biol. 37, 349-362

Article

Full-text available

Jun 1998

Homology-dependent gene silencing (HdGS) which is the generic term for transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS) and RNA-mediated virus-resistance (RmVR) has been shown to frequently occur in transgenic plants. The role of RNA as a target and initiator of PTGS and RmVR is more and more manifested. Because TGS is assumed to be induced by a DNA-DNA interaction-mediated promoter methylation, a possible involvement of RNA in TGS was not really considered up to now. In this review we attempt to demonstrate that all three types of HdGS could be triggered by one RNA-based mechanism. A model proposing TGS as a consequence of RNA-directed DNA methylation (RdDM) and a refined mRNA threshold mechanism are presented. In contrast to the view that high amounts of mRNA are required we assume that the concentration of RNAs that can serve as efficient templates for a plant-encoded RNA-directed RNA polymerase (RdRP) plays a key role in HdGS and possibly also in natural gene regulation of non-transformed cells. According to this idea a particular information must be encoded to render mRNA turn-over products a suitable RdRP substrate. It will be discussed that such a mechanism could account for the silencing phenomena of poorly transcribed transgenes. Finally, an explanation for the coherency between PTGS and DNA methylation is documented.

Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31

Article

Full-text available

May 1999

The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin.

Epigenetic inheritance at the agouti locus in the mouse

Article

Full-text available

Dec 1999

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

Silencing of Retrotransposons in Arabidopsis and Reactivation by the ddm1 Mutation

Article

Full-text available

Apr 2000

Gene silencing associated with repeated DNA sequences has been reported for many eukaryotes, including plants. However, its biological significance remains to be determined. One important function that has been proposed is the suppression of transposons. Here, we address transposon suppression by examining the behavior of the tobacco retrotransposon Tto1 and endogenous retrotransposons in Arabidopsis. After an initial increase in copy number because of active transposition in the Arabidopsis genome, Tto1 became silent. The amount of transcript was reduced, and the inactivated Tto1 became methylated. This silencing correlated with an increase in copy number. These phenomena mimic repeat-induced gene silencing. The homozygous ddm1 (for decrease in DNA methylation) mutation of Arabidopsis results in genomic DNA hypomethylation and the release of silencing in repeated genes. To investigate the role of DNA methylation and the gene-silencing machinery in the suppression of Tto1, we introduced the ddm1 mutation into an Arabidopsis line carrying inactivated Tto1 copies. In the homozygous ddm1 background, Tto1 became hypomethylated and transcriptionally and transpositionally active. In addition, one of the newly isolated endogenous Arabidopsis retrotransposon families, named Tar17, also became hypomethylated and transcriptionally active in the ddm1 mutant background. Our results suggest that the inactivation of retrotransposons and the silencing of repeated genes have mechanisms in common.

Hammond SM, Bernstein E, Beach D, Hannon GJAn RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293-296

Article

Full-text available

Apr 2000

In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals

Article

Full-text available

Nov 2000

Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Here we report the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants.

Transgene and Transposon Silencing in Chlamydomonas reinhardtii by a DEAH-Box RNA Helicase

Article

Full-text available

Dec 2000

The molecular mechanism(s) responsible for posttranscriptional gene silencing and RNA interference remain poorly understood. We have cloned a gene (Mut6) from the unicellular green alga Chlamydomonas reinhardtii that is required for the silencing of a transgene and two transposon families. Mut6 encodes a protein that is highly homologous to RNA helicases of the DEAH-box family. This protein is necessary for the degradation of certain aberrant RNAs, such as improperly processed transcripts, which are often produced by transposons and some transgenes.

Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363-366

Article

Full-text available

Feb 2001

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline

Article

Jul 2001
CURR BIOL

Background: The injection of double-stranded RNA (dsRNA) has been shown to induce a potent sequence-specific inhibition of gene function in diverse invertebrate and vertebrate species. The homology-dependent posttranscriptional gene silencing (PTGS) caused by the introduction of transgenes in plants may be mediated by dsRNA. The analysis of Caenorhabditis elegans mutants impaired with dsRNA-mediated silencing and studies in plants implicate a biological role of dsRNA-mediated silencing as a transposon-repression and antiviral mechanism. Results: We investigated the silencing of testis-expressed Stellate genes by paralogous Su(Ste) tandem repeats, which are known to be involved in the maintenance of male fertility in Drosophila melanogaster. We found that both strands of repressor Su(Ste) repeats are transcribed, producing sense and antisense RNA. The Stellate silencing is associated with the presence of short Su(Ste) RNAs. Cotransfection experiments revealed that Su(Ste) dsRNA can target and eliminate Stellate transcripts in Drosophila cell culture. The short fragment of Stellate gene that is homologous to Su(Ste) was shown to be sufficient to confer Su(Ste)-dependent silencing of a reporter construct in testes. We demonstrated that Su(Ste) dsRNA-mediated silencing affects not only Stellate expression but also the level of sense Su(Ste) RNA providing a negative autogenous regulation of Su(Ste) expression. Mutation in the spindle-E gene relieving Stellate silencing also leads to a derepression of the other genomic tandem repeats and retrotransposons in the germline. Conclusions: Homology-dependent gene silencing was shown to be used to inhibit Stellate gene expression in the D. melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated to control expression of retrotransposons in the germline.

RNA interference is required for normal centromere function in fission yeast (vol 11, pg 137, 2003)

Article

Jan 2003

Erratum: EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans (Current Biology (2000) 10 (169-178))

Article

May 2000

Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification

Article

Jan 2003

Retraction - Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing (vol 301, pg 1069, 2003)

Article

Oct 2005

Identification of Arabidopsis Histone Deacetylase HDA6 Mutants That Affect Transgene Expression

Article

May 2001

Jane Murfett

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a β-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)–selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.

Silencing of Retrotransposons in Arabidopsis and Reactivation by the ddm1 Mutation

Article

Mar 2000

Hirohiko Hirochika

Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression

Article

Apr 1990

Alexander Van der Krol

To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits.

Cellular RNA-Dependent RNA Polymerase Involved in Posttranscriptional Gene Silencing Has Two Distinct Activity Modes

Article

Jan 2002
MOL CELL

Introduction

Article

Sep 1992

Sasaki, T. , Shiohama, A. , Minoshima, S. & Shimizu, N. Identification of eight members of the Argonaute family in the human genome. Genomics 82, 323-330

Article

Sep 2003

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.

The orphanin FQ/nociceptin gene: Structure, tissue distribution of expression and functional implications obtained from knockout mice

Article

Aug 2000
PEPTIDES

Like other neuropeptides, orphanin FQ/nociceptin (OFQ/N) is encoded by a larger precursor protein. The cDNA for the OFQ/N precursor has been cloned from human, rat, mouse and bovine tissue demonstrating that this peptidergic system serves important functions that have been conserved during evolution. The structural organization of the precursor protein is similar to opioid peptide precursors, supporting the view of a common origin for the opioid systems and the OFQ/N system. In addition to OFQ/N, the precursor may encode two other biologically active peptides. Anatomic studies have revealed high levels of expression of the OFQ/N messenger RNA in brain structures involved in sensory, emotional and cognitive processing. In particular, high levels of OFQ/N mRNA were detected in the limbic system, underlining the stress attenuating activities that have been described as an important function of OFQ/N. Recently, mutant mice have been generated that lack the precursor protein of OFQ/N to further define the physiological functions of the OFQ/N system. The OFQ/N-deficient mice are characterized by an increased sensitivity to stressful stimuli and a lack of habituation to chronic and repeated stress. This review will summarize recent findings on the molecular biology of the OFQ/N precursor and relate it to possible physiological functions of this newly discovered neuropeptide system.

The regulation of developmental timing in Caenorhabditis elegans /

Article

Brenda J. Reinhart

Thesis (Ph. D., Division of Medical Sciences (Genetics))--Harvard University, 1999. Includes bibliographical references.

I transposable elements and I-R hybrid dysgenesis in Drosophila

Article

Feb 1990
TRENDS GENET

Alain Bucheton

I factors, transposable elements related to mammalian LINEs, are responsible for I-R hybrid dysgenesis in Drosophila melanogaster. Although they are not structurally related to retrovirus-like transposable elements, they appear to move around the genome via reverse transcription of a full-length RNA intermediate. The mechanism and control of this process are now being dissected at the molecular level.

Requirement for XIST in X chromosome inactivation

Article

Feb 1996

The Xist gene has been proposed as a candidate for the X inactivation centre, the master regulatory switch locus that controls X chromosome inactivation. So far this hypothesis has been supported solely by indirect evidence. Here we describe gene targeting of Xist, and provide evidence for its absolute requirement in the process of X chromosome inactivation.

Centromeres, CENP-B and Tigger too

Article

May 1997
TRENDS GENET

The highly conserved centromere-associated protein CENP-B is a common feature of mammalian centromeres. Binding sites for CENP-B, so-called 'CENP-B boxes', are present in the otherwise unrelated centromeric satellite DNAs of humans, Mus musculus, Mus caroli, ferrets, giant pandas, tree shrews and gerbils, suggesting a role for CENP-B in centromere function. However, CENP-B and its binding sites are not detected at the centromeres of mammalian Y chromosomes and few, if any, binding sites seem present on African green monkey chromosomes. There is extensive sequence similarity between CENP-B and transposase proteins encoded by the pogo superfamily of transposable elements, which includes the human Tigger elements. Intriguingly, Tigger 2 has an almost perfect match to the CENP-B-binding site within its terminal inverted repeat. Comparison of the amino acid sequence of CENP-B with related proteins raises the possibility that CENP-B might share the ability to cause single-stranded DNA breaks. Such nicks could promote recombination, as has been suggested for the Charcot-Marie-Tooth disease duplication where a recombination hotspot exists close to a mariner-like element. We suggest that by promoting nicks adjacent to CENP-B boxes, CENP-B might facilitate the evolution and maintenance of satellite sequence arrays, rather than have a direct role in centromere function.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Article

Mar 1998

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.

Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase

Article

Jun 1999

In plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. This phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. Transgene-induced gene silencing has been termed quelling in Neurospora crassa and co-suppression in plants. Quelling-defective (qde) mutants of N. crassa, in which transgene-induced gene silencing is impaired, have been isolated. Here we report the cloning of qde-1, the first cellular component of the gene-silencing mechanism to be isolated, which defines a new gene family conserved among different species including plants, animals and fungi. The qde-1 gene product is similar to an RNA-dependent RNA polymerase found in the tomato. The identification of qde-1 strongly supports models that implicate an RNA-dependent RNA polymerase in the post-transcriptional gene-silencing mechanism. The presence of qde-1 homologues in a variety of species of plants and fungi indicates that a conserved gene-silencing mechanism may exist, which could have evolved to preserve genome integrity and to protect the genome against naturally occurring transposons and viruses.

mut-7 of C. elegans, Required for Transposon Silencing and RNA Interference, Is a Homolog of Werner Syndrome Helicase and RNaseD

Article

Nov 1999
CELL

While all known natural isolates of C. elegans contain multiple copies of the Tc1 transposon, which are active in the soma, Tc1 transposition is fully silenced in the germline of many strains. We mutagenized one such silenced strain and isolated mutants in which Tc1 had been activated in the germline ("mutators"). Interestingly, many other transposons of unrelated sequence had also become active. Most of these mutants are resistant to RNA interference (RNAi). We found one of the mutated genes, mut-7, to encode a protein with homology to RNaseD. This provides support for the notion that RNAi works by dsRNA-directed, enzymatic RNA degradation. We propose a model in which MUT-7, guided by transposon-derived dsRNA, represses transposition by degrading transposon-specific messengers, thus preventing transposase production and transposition.

The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans

Article

Nov 1999
CELL

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

Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun GThe 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403: 901-906

Article

Mar 2000

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.

Ketting, R. F. & Plasterk, R. H. A genetic link between co-suppression and RNA interference in C. elegans. Nature 404, 296-298

Article

Apr 2000

Originally discovered in plants, the phenomenon of co-suppression by transgenic DNA has since been observed in many organisms from fungi to animals: introduction of transgenic copies of a gene results in reduced expression of the transgene as well as the endogenous gene. The effect depends on sequence identity between transgene and endogenous gene. Some cases of co-suppression resemble RNA interference (the experimental silencing of genes by the introduction of double-stranded RNA), as RNA seems to be both an important initiator and a target in these processes. Here we show that co-suppression in Caenorhabditis elegans is also probably mediated by RNA molecules. Both RNA interference and co-suppression have been implicated in the silencing of transposons. We now report that mutants of C. elegans that are defective in transposon silencing and RNA interference (mut-2, mut-7, mut-8 and mut-9) are in addition resistant to co-suppression. This indicates that RNA interference and co-suppression in C. elegans may be mediated at least in part by the same molecular machinery, possibly through RNA-guided degradation of messenger RNA molecules.

Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis

Article

Jun 2000
CURR BIOL

Proteins such as HP1, found in fruit flies and mammals, and Swi6, its fission yeast homologue, carry a chromodomain (CD) and a chromo shadow domain (CSD). These proteins are required to form functional transcriptionally silent centromeric chromatin, and their mutation leads to chromosome segregation defects. CSDs have only been found in tandem in proteins containing the related CD. Most HP1-interacting proteins have been found to associate through the CSD and many of these ligands contain a conserved pentapeptide motif. The 1.9 A crystal structure of the Swi6 CSD is presented here. This reveals a novel dimeric structure that is distinct from the previously reported monomeric nuclear magnetic resonance (NMR) structure of the CD from the mouse modifier 1 protein (MoMOD1, also known as HP1beta or M31). A prominent pit with a non-polar base is generated at the dimer interface, and is commensurate with binding an extended pentapeptide motif. Sequence alignments based on this structure highlight differences between CDs and CSDs that are superimposed on a common structural core. The analyses also revealed a previously unrecognised circumferential hydrophobic sash around the surface of the CD structure. Dimerisation through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites.

An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus

Article

Jun 2000
CELL

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance

Article

Jun 2000
CELL

Posttranscriptional gene silencing (PTGS) in plants resuits from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, definitively proving that PTGS is an antiviral defense mechanism that can also target transgene RNA for degradation.

Mette, M.F., Aufsatz, W., van der Winden, J., Matzke, M.A. & Matzke, A.J. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19, 5194-5204

Article

Nov 2000

Double-stranded RNA induces a post-transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double-stranded RNA containing promoter sequences. Similar to the double-stranded RNA involved in RNAi, this promoter double-stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs approximately 23 nucleotides in length. Both transcriptional and post-transcriptional gene silencing can thus be initiated by double-stranded RNAs that enter the same degradation pathway. The results also implicate double-stranded RNA in directing DNA methylation. Different constructs designed to produce double-stranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis. RNA hairpins transcribed from inverted DNA repeats were the most effective trans-acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain

Article

Apr 2001

Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.

Lachner, M., O'Carrol, D., Rea, S., Mechtler, K. & Jenuwein, T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-120

Article

Apr 2001

Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to histone modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programmes are to proceed unperturbed. Here we show that mammalian methyltransferases that selectively methylate histone H3 on lysine 9 (Suv39h HMTases) generate a binding site for HP1 proteins--a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromo domain; thus, the HP1 chromo domain is a specific interaction motif for the methyl epitope on lysine9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active SWUV39H1 HMTase. Our data define a molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin.

Whitelaw, E. & Martin, D.I. Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat. Genet. 27, 361-365

Article

May 2001

Phenotypic variation in mammals is frequently attributed to the action of quantitative trait loci (QTL) or the environment, but may also be epigenetic in origin. Here we consider a mechanism for phenotypic variation based on interference of transcription by somatically active retrotransposons. Transcriptionally competent retrotransposons may number in the tens of thousands in mammalian genomes. We propose that silencing of retrotransposons occurs by cosuppression during early embryogenesis, but that this process is imperfect and produces a mosaic pattern of retrotransposon expression in somatic cells. Transcriptional interference by active retrotransposons perturbs expression of neighboring genes in somatic cells, in a mosaic pattern corresponding to activity of each retrotransposon. The epigenotype of retrotransposon activity is reset in each generation, but incomplete resetting can lead to heritable epigenetic effects. The stochastic nature of retrotransposon activity, and the very large number of genes that may be affected, produce subtle phenotypic variations even between genetically identical individuals, which may affect disease risk and be heritable in a non-mendelian fashion.

Identification of Arabidopsis Histone Deacetylase HDA6 Mutants That Affect Transgene Expression

Article

Jun 2001

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Article

Jun 2001

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires MetI for maintenance

Article

Jun 2001
CURR BIOL

The association between DNA methylation and gene silencing has long been recognized; however, signals that initiate de novo methylation are largely unknown. In plants, recognition of RNAs that are inducers of posttranscriptional gene silencing (PTGS) can result in sequence-specific DNA methylation, and the aim of this work was to investigate whether heritable epigenetic changes can occur by this mechanism and if the Met1 methyltransferase is required. RNA-directed DNA methylation (RdDM) was initiated in 35S-GFP transgenic plants following infection with plant RNA viruses modified to carry portions of either the 35S promoter or the GFP coding region. Targeting of the promoter sequence resulted in both methylation and transcriptional gene silencing (TGS) that was inherited independently of the RNA trigger. Targeting the coding region also resulted in methylation; however, this was not inherited. Expression of Met1 was suppressed in order to investigate its role in initiation and maintenance of RdDM. Initiation of RdDM was found to be Met1-independent, whereas maintenance of methylation and TGS in the subsequent generations in the absence of the RNA trigger was Met1-dependent. Maintenance of methylation associated with systemic PTGS was also found to be Met1-independent. RNA-triggered events can lead to heritable changes in gene expression, and it is possible that initiation of other epigenetic phenomena such as trans-silencing and paramutation may have an RNA component.

Almeida, R and Allshire, RC. RNA silencing and genome regulation. Trends Cell Biol 15: 251-258

Abstract

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