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.

Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class

Science ◽  
2006 ◽  
Vol 315 (5809) ◽  
pp. 244-247 ◽  
Author(s):  
Titia Sijen ◽  
Florian A. Steiner ◽  
Karen L. Thijssen ◽  
Ronald H. A. Plasterk
Keyword(s):  
Rna Interference ◽  
Small Rnas ◽  
Rna Synthesis ◽  
Argonaute Protein ◽  
Single Nucleotide ◽  
Distinct Class ◽  
C Elegans ◽  
Secondary Sirnas ◽  
Secondary Sirna ◽  
Rnai Response

In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non–RISC (RNA-induced silencing complex)–cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5′ di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.

scholarly journals Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

2018 ◽  
Author(s):  
Snusha Ravikumar ◽  
Sindhuja Devanapally ◽  
Antony M Jose
Keyword(s):  
Rna Interference ◽  
Small Rnas ◽  
Target Genes ◽  
Somatic Cells ◽  
Cell Types ◽  
Random Sets ◽  
Multiple Sources ◽  
Double Stranded Rna ◽  
C Elegans ◽  
A Cell

ABSTRACTDelivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode C. elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we analyze instances of silencing in somatic cells that lack RRF-1. By varying dsRNA sources and target genes, we find that silencing in the absence of RRF-1 is most obvious when dsRNA from neurons is used to silence genes in intestinal cells. This silencing requires EGO-1, but the lineal identity of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. As a result, all cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against the use of a few molecules as proxies for predicting the behavior of a cell.Graphical AbstractRandom sets of cells can either receive different amounts of double-stranded RNA from neurons or use different RdRPs – RRF-1 only versus RRF-1 or EGO-1 – to perform the same function.

scholarly journals E. coli OxyS non-coding RNA does not trigger RNAi in C. elegans

2014 ◽  
Author(s):  
Alper Akay ◽  
Peter Sarkies ◽  
Eric Alexander Miska
Keyword(s):  
Small Rnas ◽  
Biological Function ◽  
Rapid Development ◽  
Rnai Pathway ◽  
Regulate Gene Expression ◽  
Double Stranded Rna ◽  
E Coli ◽  
C Elegans ◽  
Non Coding Rna ◽  
Rnai Response

The discovery of RNA interference (RNAi) in C. elegans has had a major impact on scientific research, led to the rapid development of RNAi tools and has inspired RNA-based therapeutics. Astonishingly, nematodes, planaria and many insects take up double-stranded RNA (dsRNA) from their environment to elicit RNAi; the biological function of this mechanism is unclear. Recently, the E. coli OxyS non-coding RNA was shown to regulate gene expression in C. elegans when E. coli is offered as food. This was surprising given that C. elegans is unlikely to encounter E. coli in nature. To directly test the hypothesis that the E. coli OxyS non-coding RNA triggers the C. elegans RNAi pathway, we sequenced small RNAs from C. elegans after feeding with bacteria. We clearly demonstrate that the OxyS non-coding RNA does not trigger an RNAi response in C. elegans. We conclude that the biology of environmental RNAi remains to be discovered.

scholarly journals Intronic MicroRNA (miRNA)

2006 ◽  
Vol 2006 ◽  
pp. 1-13 ◽  
Author(s):  
Shi-Lung Lin ◽  
Joseph D. Miller ◽  
Shao-Yao Ying
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Regulatory System ◽  
Fine Tuning ◽  
Messenger Rnas ◽  
Noncoding Dna ◽  
Protein Coding ◽  
C Elegans ◽  
Adult Mice

Nearly 97% of the human genome is composed of noncoding DNA, which varies from one species to another. Changes in these sequences often manifest themselves in clinical and circumstantial malfunction. Numerous genes in these non-protein-coding regions encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) with complete or partial complementarity, are useful for the design of new therapies against cancer polymorphisms and viral mutations. Currently, many varieties of miRNA are widely reported in plants, animals, and even microbes. Intron-derived microRNA (Id-miRNA) is a new class of miRNA derived from the processing of gene introns. The intronic miRNA requires type-II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of Id-miRNA have been identified inC elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in not only human and mouse cells, but in also zebrafish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of intron-mediated gene silencing via functional miRNA in cell and in vivo. These findings suggest an intracellular miRNA-mediated gene regulatory system, fine-tuning the degradation of protein-coding messenger RNAs.

Viroid-induced DNA methylation in plants

2013 ◽  
Vol 4 (6) ◽  
pp. 557-565 ◽  
Author(s):  
Athanasios Dalakouras ◽  
Elena Dadami ◽  
Michael Wassenegger
Keyword(s):  
Dna Methylation ◽  
Small Rnas ◽  
Rna Silencing ◽  
De Novo ◽  
Rna Degradation ◽  
Double Stranded Rna ◽  
Methyl Group ◽  
Rna Molecules ◽  
Cumulative Data

AbstractIn eukaryotes, DNA methylation refers to the addition of a methyl group to the fifth atom in the six-atom ring of cytosine residues. At least in plants, DNA regions that become de novo methylated can be defined by homologous RNA molecules in a process termed RNA-directed DNA methylation (RdDM). RdDM was first discovered in viroid-infected plants. Viroids are pathogenic circular, non-coding, single-stranded RNA molecules. Members of the Pospiviroidae family replicate in the nucleus through double-stranded RNA intermediates, attracting the host RNA silencing machinery. The recruitment of this machinery results in the production of viroid-derived small RNAs (vd-sRNAs) that mediate RNA degradation and DNA methylation of cognate sequences. Here, we provide an overview of the cumulative data on the field of viroid-induced RdDM and discuss three possible scenarios concerning the mechanistic details of its establishment.

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 De NovoRNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase

2016 ◽  
Vol 36 (8) ◽  
pp. 1248-1259 ◽  
Author(s):  
Yoshiko Maida ◽  
Mami Yasukawa ◽  
Kenkichi Masutomi
Keyword(s):  
Rna Polymerase ◽  
Reverse Transcriptase ◽  
De Novo ◽  
Telomerase Reverse Transcriptase ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Human Carcinoma ◽  
Content Type ◽  
Secondary Sirnas

RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such asCaenorhabditis elegansandNeurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAsde novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However,de novosynthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5′-triphosphorylated ends, which indicatesde novosynthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity.

scholarly journals Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain

2021 ◽  
Author(s):  
Shirui Chen ◽  
Wei Liu ◽  
Masahiro Naganuma ◽  
Yukihide Tomari ◽  
Hiro-oki Iwakawa
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Functional Differentiation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Secondary Sirnas ◽  
Fixed End ◽  
Paz Domain ◽  
Reproductive Processes

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt heterochromatic small interfering RNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs). The former small RNAs are linked to plant heterochromatin, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient "eudicot-type" DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 3′ overhangs and 5′ phosphates, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5 which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

scholarly journals RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
Hanna Lukas ◽  
Maria Mendez-Lago ◽  
René F. Ketting
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Cloning Efficiency ◽  
Library Preparation ◽  
Small Interfering Rnas ◽  
Rdrp Activity ◽  
C Elegans ◽  
Rna Pathways ◽  
Small Rna Species

AbstractRNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.

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.

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