scholarly journals Each cell needs long double-stranded RNA for silencing by feeding RNAi in C. elegans

2016 ◽  
Author(s):  
Pravrutha Raman ◽  
Soriayah M Zaghab ◽  
Edward C Traver ◽  
Antony M Jose
Keyword(s):  
Rna Interference ◽  
Single Copy ◽  
Animal Cells ◽  
Tissue Specific ◽  
Double Stranded Rna ◽  
C Elegans ◽  
Dsrna Binding ◽  
Dsrna Binding Protein ◽  
Systemic Rnai ◽  
Derivatives Of

ABSTRACTLong double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm C. elegans, where it is thought that derivatives of ingested dsRNA, including short dsRNAs, move between cells and cause systemic silencing. Movement of short dsRNAs has been inferred using tissue-specific rescue of the long dsRNA-binding protein RDE-4 by expressing it from repetitive transgenes. We found that the use of repetitive transgenes for the tissue-specific rescue of a gene could inhibit RNAi within that tissue and could result in misexpression of the gene in other tissues. Both inhibition and misexpression were not detectable when a single-copy transgene was used for tissue-specific rescue. In animals with single-copy rescue of RDE-4, RNAi was restricted to the tissue with RDE-4 expression. Thus, unlike previous observations using repetitive transgenes, these results suggest that binding of long dsRNA by RDE-4 in each silenced cell is required for systemic RNAi. Taken together with the requirement for long dsRNA to trigger RNAi in insects, these results suggest that the entry of long dsRNA is a necessary first step for feeding RNAi in animal cells.

scholarly journals Inducible Systemic RNA Silencing in Caenorhabditis elegans

2003 ◽  
Vol 14 (7) ◽  
pp. 2972-2983 ◽  
Author(s):  
Lisa Timmons ◽  
Hiroaki Tabara ◽  
Craig C. Mello ◽  
Andrew Z. Fire
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Normal Animal ◽  
Cell Types ◽  
Animal Cells ◽  
Tissue Specific ◽  
C Elegans ◽  
Systemic Rna

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

scholarly journals Double-Stranded-RNA-Binding Protein 2 Participates in Antiviral Defense

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Károly Fátyol ◽  
Katalin Anna Fekete ◽  
Márta Ludman
Keyword(s):  
Rna Interference ◽  
Virus Infection ◽  
Nicotiana Benthamiana ◽  
Rna Binding ◽  
Binding Protein ◽  
Systemic Necrosis ◽  
Double Stranded Rna ◽  
Content Type ◽  
Dsrna Binding ◽  
Dsrna Binding Protein

ABSTRACT Double-stranded RNA (dsRNA) is a common pattern formed during the replication of both RNA and DNA viruses. Perception of virus-derived dsRNAs by specialized receptor molecules leads to the activation of various antiviral measures. In plants, these defensive processes include the adaptive RNA interference (RNAi) pathway and innate pattern-triggered immune (PTI) responses. While details of the former process have been well established in recent years, the latter are still only partially understood at the molecular level. Nonetheless, emerging data suggest extensive cross talk between the different antiviral mechanisms. Here, we demonstrate that dsRNA-binding protein 2 (DRB2) of Nicotiana benthamiana plays a direct role in potato virus X (PVX)-elicited systemic necrosis. These results establish that DRB2, a known component of RNAi, is also involved in a virus-induced PTI response. In addition, our findings suggest that RNA-dependent polymerase 6 (RDR6)-dependent dsRNAs play an important role in the triggering of PVX-induced systemic necrosis. Based on our data, a model is formulated whereby competition between different DRB proteins for virus-derived dsRNAs helps establish the dominant antiviral pathways that are activated in response to virus infection. IMPORTANCE Plants employ multiple defense mechanisms to restrict viral infections, among which RNA interference is the best understood. The activation of innate immunity often leads to both local and systemic necrotic responses, which confine the virus to the infected cells and can also provide resistance to distal, noninfected parts of the organism. Systemic necrosis, which is regarded as a special form of the local hypersensitive response, results in necrosis of the apical stem region, usually causing the death of the plant. Here, we provide evidence that the dsRNA-binding protein 2 of Nicotiana benthamiana plays an important role in virus-induced systemic necrosis. Our findings are not only compatible with the recent hypothesis that DRB proteins act as viral invasion sensors but also extends it by proposing that DRBs play a critical role in establishing the dominant antiviral measures that are triggered during virus infection.

RNA interference in Caenorhabditis elegans: Uptake, mechanism, and regulation

Parasitology ◽  
2011 ◽  
Vol 139 (5) ◽  
pp. 560-573 ◽  
Author(s):  
JIMMY J. ZHUANG ◽  
CRAIG P. HUNTER
Keyword(s):  
Rna Interference ◽  
Genetic Analysis ◽  
Large Scale ◽  
Parasitic Nematodes ◽  
Loss Of Function ◽  
Uptake Mechanism ◽  
Delivery Methods ◽  
C Elegans ◽  
Systemic Rnai ◽  
Powerful Research Tool

SUMMARYRNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.

scholarly journals New strains for tissue-specific RNAi studies in Caenorhabditis elegans

2018 ◽  
Author(s):  
Jason S. Watts ◽  
Henry F. Harrison ◽  
Shizue Omi ◽  
Quentin Guenthers ◽  
James Dalelio ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Caenorhabditis Elegans ◽  
Gene Function ◽  
Target Genes ◽  
Resistant Mutant ◽  
Knockout Mutant ◽  
Tissue Specific ◽  
Double Stranded Rna ◽  
C Elegans ◽  
Insight Into

AbstractRNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

scholarly journals Single-copy Knock-In Loci for Defined Gene Expression in C. elegans

2018 ◽  
Author(s):  
Carlos G Silva-Garcia ◽  
Caroline Heintz ◽  
Sneha Dutta ◽  
Nicole M Clark ◽  
Anne Lanjuin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Cdna Sequence ◽  
Single Copy ◽  
Tissue Specific ◽  
C Elegans

We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any cDNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any cDNA sequence can be introduced as a single integrated copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical and effective approach for generating single-copy non-silenced transgenes in C. elegans.

scholarly journals The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1, and a DExH-Box Helicase to Direct RNAi in C. elegans

Cell ◽  
2002 ◽  
Vol 109 (7) ◽  
pp. 861-871 ◽  
Author(s):  
Hiroaki Tabara ◽  
Erbay Yigit ◽  
Haruhiko Siomi ◽  
Craig C. Mello
Keyword(s):  
Binding Protein ◽  
C Elegans ◽  
Dsrna Binding ◽  
Dsrna Binding Protein

Distinct Populations of Primary and Secondary Effectors During RNAi in C. elegans

Science ◽  
2006 ◽  
Vol 315 (5809) ◽  
pp. 241-244 ◽  
Author(s):  
Julia Pak ◽  
Andrew Fire
Keyword(s):  
Rna Interference ◽  
Small Rnas ◽  
De Novo ◽  
Messenger Rnas ◽  
Cellular Regulation ◽  
Rdrp Activity ◽  
Antisense Transcripts ◽  
Double Stranded Rna ◽  
C Elegans ◽  
Secondary Sirnas

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: “Primary siRNAs” (derived from DICER nuclease-mediated cleavage of the original trigger) and “secondary siRNAs” [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

scholarly journals Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response

2005 ◽  
Vol 79 (17) ◽  
pp. 11105-11114 ◽  
Author(s):  
Joao T. Marques ◽  
Dominique Rebouillat ◽  
Chilakamarti V. Ramana ◽  
Junko Murakami ◽  
Jason E. Hill ◽  
...  
Keyword(s):  
Suppressor Gene ◽  
Encephalomyocarditis Virus ◽  
Down Regulation ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Dsrna Binding Protein ◽  
Infected Cells ◽  
Induced Apoptosis ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACT p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.

scholarly journals RNAi targeting Caenorhabditis elegans α-arrestins has little effect on lifespan

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1515
Author(s):  
Sangsoon Park ◽  
Yoonji Jung ◽  
Seon Woo A. An ◽  
Heehwa G. Son ◽  
Wooseon Hwang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Functional Role ◽  
Future Research ◽  
Biological Processes ◽  
Receptor Desensitization ◽  
Double Stranded Rna ◽  
C Elegans

Background: α-arrestins are a family of proteins that are implicated in multiple biological processes, including metabolism and receptor desensitization. Methods: Here, we sought to examine the roles of α-arrestins in the longevity of Caenorhabditis elegans through an RNA interference screen. Results: We found that feeding worms with bacteria expressing double-stranded RNA against each of 24 out of total 29 C. elegans α-arrestins had little effect on lifespan. Thus, individual C. elegans α-arrestins may have minor effects on longevity. Conclusions: This study will provide useful information for future research on the functional role of α-arrestins in aging and longevity.

A Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit for tissue specific profiling of actively translated mRNAs in C. elegans.

2021 ◽  
Author(s):  
Laura E Wester ◽  
Anne Lanjuin ◽  
Emanuel H W Bruckisch ◽  
Maria C Perez Matos ◽  
Caroline Heintz ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Single Copy ◽  
Specific Cell ◽  
Cell Type ◽  
Specific Expression ◽  
Tissue Specific ◽  
C Elegans ◽  
Cell Type Specific

Translating Ribosome Affinity Purification (TRAP) methods have emerged as a powerful approach to profile actively translated transcripts in specific cell and tissue types. Epitope tagged ribosomal subunits are expressed in defined cell populations and used to pull down ribosomes and their associated mRNAs, providing a snapshot of cell type-specific translation occurring in that space and time. Current TRAP toolkits available to the C. elegans community have been built using multi-copy arrays, randomly integrated in the genome. Here we introduce a Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit, a collection of C. elegans strains engineered by CRISPR in which tissue specific expression of FLAG tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. In depth characterization of the SKI TRIP strains and methodology shows that 3xFLAG tagged RPL-22 expressed from its endogenous locus or within defined cell types incorporates into actively translating ribosomes and can be used to efficiently and cleanly pull-down cell type specific transcripts without impacting overall mRNA translation or fitness of the animal. We propose SKI TRIP use for the study of processes that are acutely sensitive to changes in translation, such as aging.

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