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RNA Biology

ISSN: 1547-6286 (Print) 1555-8584 (Online) Journal homepage: http://www.tandfonline.com/loi/krnb20

Artificial and natural RNA interactions betweenbacteria and C. elegans

Fabian Braukmann, David Jordan & Eric Miska

To cite this article: Fabian Braukmann, David Jordan & Eric Miska (2017) Artificial andnatural RNA interactions between bacteria and C.�elegans, RNA Biology, 14:4, 415-420, DOI:10.1080/15476286.2017.1297912

To link to this article: https://doi.org/10.1080/15476286.2017.1297912

Accepted author version posted online: 23Mar 2017.Published online: 05 Apr 2017.

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REVIEW

Artificial and natural RNA interactions between bacteria and C. elegans

Fabian Braukmann a,b, David Jordana,b, and Eric Miskaa,b,c

aGurdon Institute, University of Cambridge, Cambridge, UK; bDepartment of Genetics, University of Cambridge, Cambridge, UK; cWellcome Trust SangerInstitute, Wellcome Trust Genome Campus, Cambridge, UK

ARTICLE HISTORYReceived 20 December 2016Revised 13 February 2017Accepted 17 February 2017

ABSTRACTNineteen years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans inan experimental setting48 the biologic role of this trans-kingdom RNA-based communication remainsunknown. Here we summarize our current understanding on the mechanism and potential role of suchsocial RNA. KEYWORDS

Bacteria; C. elegans;environmental RNAi; mobileRNA

Introduction

Caenorhabditis elegans is a small (�1 mm) free-livingnematode found in microbe rich rotting fruits and vegetationin temperate climates. It was chosen by Sydney Brenner as amodel organism due to its numerous advantages for geneticsand cell biologic analysis. It is easy to maintain in the labora-tory, living on a diet of Escherichia coli bacteria. It is a self-fer-tilizing hermaphrodite with a relatively short generation time(3 days), and a large brood size (�300), facilitating the genera-tion of large isogenic populations of worms. In addition, it istransparent, an advantage for microscopy and cell biology.Through the painstaking work of John Sulston, together withRobert Horvitz, the entire cell lineage from egg to adult wasmapped, including the 302 neuron nervous system.44,45

Discovery of RNA interference (RNAi) in an animal

The use of RNA sequences which are complementary (anti-sense) to some region of the mRNA of a gene of interest, inhopes that through Watson-Crick base pairing, the 2 molculeswill hybridize and lead to misregulation of the expression ofthat gene, was first described by Izant and Weintraub in 198419

as an alternative to classical genetic analysis of mutants. Anti-sense RNA continued to be developed and became a commontool in the molecular biology toolkit.8 However, antisense geneknockdowns were generally not robust, and the mechanism bywhich silencing occurred was not known. It was not until adecade later that the first tantalizing hints of the pathway, laterto become known as the RNA interference, or RNAi pathway,were uncovered. In 1995, Guo and Kemphures, in an attemptto inject antisense RNA to knockdown par-1 in C. elegans,reported similar knockdown efficiency for both the antisenseand the sense strands alone, as well as the co-injection of boththe sense and antisense strands corresponding to the par-1mRNA.15 While previous reports of so-called co-suppression,

or quelling, had been reported in petunias,34 and in Neosporacrassa,37 this was the first example in an animal, and the inspi-ration for the seminal work of Fire and Mello, who, in 1998,hypothesized that contaminants in the single-stranded RNA(ssRNA) preparation led to double-stranded RNA (dsRNA)and that these dsRNAs were responsible for the silencing. Theirwork showed that targets could be efficiently silenced by injec-tion of dsRNAs with sequence complementarity to the target.9

RNAi mechanism in C. elegans

The RNAi pathway is used to regulate gene expression.11 Thepathway begins with the processing of either an endogenouslygenerated or an environmentally supplied dsRNA. The dsRNAbinding protein RDE-4 interacts with the dsRNA and initiatescleavage of the RNA by the conserved endonucleaseDCR-1.36,47 DCR-1 produces double stranded-short interferingRNAs (ds-siRNAs) which are subsequently loaded into theArgonaute protein RDE-1.14,53 The passenger strand isdegraded and the remaining strand guides RDE-1 via Watsonand Crick base pairing to a target mRNA.43 The mRNA-RDE-1complex is thought to recruit the RNA-dependent RNA poly-merase (RdRP) RRF-1 which leads to generation of secondarysiRNAs.35,41 The secondary siRNAs are loaded into other pro-teins of the Argonaute family and initiate post-transcriptionalgene silencing in the cytoplasm via mRNA degradation and co-transcriptional gene silencing in the nucleus via modification ofthe chromatin.12,13

Systemic and environmental RNAi

While it took many years for the details described above to beworked out, soon after RNAi was first described in 1998, Tab-ara and Mello showed that RNAi could be initiated by soakingworms in a solution of dsRNA,46 and Timmons and Fire dem-onstrated that by expressing dsRNAs in the E. coli food of C.

CONTACT Eric Miska eam29@cam.ac.uk The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.© 2017 Taylor & Francis Group, LLC

RNA BIOLOGY2017, VOL. 14, NO. 4, 415–420http://dx.doi.org/10.1080/15476286.2017.1297912

elegans, silencing could be induced by feeding.49 This hasbecome known as environmental RNAi. The RNAi by feedingtechnique was optimized a few years later, using RNase defi-cient bacteria,26 and a few years after that, the first genomewide RNAi screen was reported.25 In the intervening year,working to understand how dsRNA injected into one tissuecould lead to silencing in other tissues, Winston and Hunterreported the results of the first genetic screens for this so-calledsystemic RNAi, uncovering systemic RNAi deficient (Sid)genes, including sid-1,51 which encodes a dsRNA specific trans-membrane channel.7 A second Sid protein, SID-2, which is asingle pass trans-membrane protein, was found to be essentialfor environmental RNAi (See detailed review24). The systemicRNAi genes are distinct from the genes involved in the canoni-cal RNAi pathway described above and are not required forRNAi itself. In other words, in the absence of systemic RNAigenes, silencing still occurs, but it is localized and must be initi-ated by endogenously produced or injected dsRNA. The genesinvolved in systemic RNAi can be divided into 2 classes. Oneset of genes are important for the uptake of dsRNA from theenvironment into the worm, while a second set of genes areimportant for the transport of RNA, which is already in theworm, from one tissue to another.

Environmental RNAi

In the current understanding of the environmental RNAi path-way, dsRNA from the environment is initially internalizedfrom the gut lumen. Subsequently, the internalized dsRNA iseither transported through the intestinal cell from the apical tothe basal surface and released into the pseudocoelom, or is firstreleased into the gut cytoplasm and from there exported intothe pseudocoelom. The dsRNA is later imported into the recipi-ent tissue, where it enters the RNAi pathway.24

SID-2In C. elegans, the only known role of the single-pass trans-membrane protein SID-2 is the uptake of dsRNA from theenvironment into the gut (Fig. 1A). Sid-2 mutants do not showany sign of RNAi by feeding, but are able to down-regulateGFP in the body wall via pharyngeal expressed RNA hairpinswhich target GFP (hpGFP).52 Therefore SID-2 is not requiredfor intercellular transport of RNA within a C. elegans. However,SID-2 is required for dsRNA uptake from the gut lumen intothe worm as indicated by the failure to detect fluorescentlylabeled dsRNA inside sid-2 mutant worms after soaking.31 Inaddition, SID-2 localizes in the gut as indicated by expressionanalysis ofSID-2::GFP fusion protein.52 Expression of sid-2 in a heterolo-gous system showed specificity for the uptake of � 50 bpdsRNA.31 These findings indicate SID-2s important role inenvironmental RNAi, and in the future, studies on naturallyoccurring RNA uptake should focus on dsRNA uptake in thegut. However, it cannot be ruled out that another mechanismof RNA uptake exists that does not require SID-2. Such a sys-tem would have different properties and if it exists, its functionmay be independent of the RNAi pathway. Here, we focus onthe potential of environmental dsRNA as a mechanism ofcommunication.

Systemic RNAi

SID-1The first gene identified to be important for dsRNA transportwas sid-1.51 SID-1 is a multispan trans-membrane protein andputative dsRNA channel. Expression of a SID-1::GFP fusionprotein showed localization at the plasma membrane of almostall C. elegans cells, but appeared to be excluded from most neu-rons.51 The use of sid-1 mutant worms expressing SID-1 in amosaic fashion indicated that SID-1 is required for the importof mobile RNA (Fig. 1B).21,22 Recent experiments have shedfurther light on the precise mechanism of dsRNA import viaSID-1. This year, Marr�e and colleagues showed that fluores-cently labeled dsRNA enters a cell via RME-2 (Receptor Medi-ated Endocytosis), a member of the low-density lipoproteinreceptor superfamily, but that SID-1 is required for the releaseof the dsRNA into the cytoplasm (Fig. 1E).30 Work in Drosoph-ila melanogaster S2 cells indicated that SID-1 enhances theuptake of dsRNA longer than 50 bp, but that completesequence complementarity is not required. Thus primary miR-NAs, which include a hairpin, can potentially be transported bySID-1.42 Surprisingly, neurons are deficient in RNAi by feeding,however overexpression of SID-1 from a neuronal promoterrenders these cells RNAi competent indicating that SID-1 isrequired to take up dsRNA into neurons.4 These findings sug-gest that naturally occurring dsRNA can regulate the functionof most tissues except for neurons. Therefore, an environmen-tal dsRNA can have a systemic effect on gene expression oreven a transgenerational effect via the germline. Current evi-dence suggests that SID-1 and SID-2 have similar physicalrequirements for the transport of RNA.

SID-3SID-3 is a conserved tyrosine kinase required for the import ofmobile RNAs (Fig. 1C).23 SID-3 is expressed in many tissues inthe cytoplasm in punctuated foci as indicated by the analysis ofthe expression of sid-3::gfp and sid- 3::mCherry transgenes. It isspeculated that these foci are related to endocytosis, since thehuman homolog of SID-3, the activated CDC42-associatedkinase (ACK) localizes to endocytic vesicles.16 The use of sid-3mutant worms with tissue specific rescue of sid-3 revealed thatSID-3 is required for the import of mobile RNA.23 It is believedthat SID-3 is an important protein in a signaling pathway forefficient RNA import, since the human CDC42 is implicated inmany signaling pathways.32 Interestingly, in the absence ofSID-3, local RNAi is enhanced.

SID-5SID-5 is a predicted single-pass trans membrane proteinimportant for efficient systemic RNAi. SID-5 is an importantmember of the pathway because it links RNA mobility to vesi-cle transport. Reports previously indicated that endosomal pro-teins are required for efficient RNAi but not for RNAmobility.28 SID-5 is the first protein of the Sid-pathway, whichco-localizes with endosome markers and has a demonstratedrole in RNA transport.17 SID-5 is required for RNAi in the gutvia RNAi by feeding, suggesting that SID-5 has a similar role asSID-2. However, immunofluorescent stains show SID-5 expres-sion in a range of tissues, in contrast to SID-2 which is

416 F. BRAUKMANN ET AL.

expressed only in the gut. In addition, SID-5 is relevant forRNA silencing in the body wall muscle (bwm) initiated fromthe pharynx in contrast to SID-2. This indicates that SID-5 hasa different role than SID-2. In addition, SID-5s role must differfrom the role of SID-1 and SID-3, as a rescue of sid-5 in therecipient tissue (bwm) does not restore RNA silencing.17 Thisindicates that it is important for the export of RNA (Fig. 1C).Further experiments are required to determine the exact role ofSID-5 in the systemic RNAi pathway.

SEC-22SEC-22 is a SNARE (Soluble NSF Attachment Protein Recep-tor) protein. SNAREs are a superfamily of proteins involved inmembrane fusion and therefore important for vesicle traffick-ing.50 In C. elegans, SEC-22 suppresses small RNA mediatedsilencing in a sid-5 dependent manner. The use of sid-5 mutantworms with tissue specific rescue of sec-22 indicates that SEC-22 either inhibits trafficking or the RNAi machinery in the cell(Fig. 1C). In addition, mCherry::SEC-22 fusion proteins localizeto the late endosome similar to SID-5. Furthermore, SEC-22interacts with SID-5 in a yeast 2-hybrid system.54 Therefore, itis clear that SEC-22 is a negative regulator of RNAi and onemay speculate that SEC-22 does so by promoting late endo-some degradation. However, further experiments are requiredto untangle the role of SEC-22 in systemic RNAi or regulationof the RNAi factors within the cell.

RSD-3RNAi spreading defective-3 (rsd-3) is required for RNA uptakein somatic and germline cells.18 Similar to SID-1 and SID-3,RSD-3 functions in RNA import (Fig. 1C). RSD-3 encodes fora homolog of epsinR and has a conserved ENTH (epsin N-ter-minal homology) domain. In mammalian systems, the ENTHdomain is important for endomembrane trafficking.39 Interest-ingly, the ENTH domain of RSD-3 is sufficient in C. elegans formobile RNAi. This suggests that the ENTH domain is able tomediate on its own downstream activities for RNA uptake oralternatively the ENTH domain is sufficient to down-regulate aRNA uptake suppressing signal. This provides another piece ofevidence for the importance of the vesicle transport pathwaysfor mobile RNA. Transgene expression experiments show thatRSD-3 is expressed in many tissues and co-localizes with fac-tors of the trans-Golgi network and the endosome.18 While theexact role of RSD-3 remains unclear, it is tempting to speculateabout its role, and the role of its ENTH domain is to functionas cargo adaptor. However, the ENTH domain has not beenreported to bind RNA, therefore it is unlikely that RSD-3directly interacts with RNA. More likely, the role of rsd-3 couldbe to sort other SID pathway members to their correct locationsin vesicle trafficking.

MUT-2Mut-2, known also as rde-3, is a putative nucleotidyltransferaseimportant for mobile RNA. Mut-2 was first identified to play arole in transposon regulation.6 Subsequently, it became evidentthat mut-2 is essential for RNAi induced by feeding, but is notrequired for RNAi induced by transgene-driven expression ofdsRNA.5 A more detailed analysis by 22 found that mut-2 isrequired for silencing by transgene-driven expression of

dsRNA that is spatially separated from the tissue where thesilencing occurs. Surprisingly, mut-2 can be rescued by theexpression of mut-2 in only the tissue producing the mobileRNA (donor), or in only the tissue in which the silencingoccurs (recipient). This indicates that mut-2 has a dual role (1)exporting the RNAi signal from the donor tissue, and (2) gener-ating efficient RNAi in the recipient tissue (Fig. 1D). Therefore,in the donor tissue, the role of MUT-2 could be marking themobile RNA by nucleotide addition for efficient export, and inthe recipient, to generate efficiently the secondary siRNA.

Summary of environmental RNAi

Overall, environmental RNAi is an intriguing biologic process.In the last 20 years, we have gained insights into the mecha-nism via genetic analysis and have come to understand some ofthe physical requirements for RNA mobility in C. elegans. RNAtransport is not as simple as diffusion and uptake in a recipienttissue. Many cellular processes, such as vesicle transport, areinvolved to regulate export and import of mobile RNAs. How-ever, we still do not understand how RNA transport is regu-lated and how an RNA is chosen for delivery. Furthermore, ourunderstanding of RNA transport remains very limited to itsfunction in relation to RNAi, additional functions remain to beexplored.

From artificial to natural context

RNAi by feeding has become a simple and powerful tool for thespecific knockdown of genes. That it works so well, however, isintriguing because so far, it is only known in the artificial con-text of bacteria genetically engineered to express dsRNA withsequence complementarity to endogenous C. elegans genes.This leads one to speculate whether there may be a natural con-text in which the uptake of RNA for silencing may be impor-tant. While there are clear examples of trans-kingdomcommunication via RNA intermediates in a natural context,e.g. between humans and some of their parasites, and betweenplants and fungi,27 it is odd that no natural context has yetbeen discovered for C. elegans, where RNAi by feeding is such apowerful tool. Some recent experiments by Melo and Ruvkunmay provide a conceptual bridge between artificial and naturalRNAi by feeding. In these experiments, Melo and colleaguesshowed that E. coli artificially expressing dsRNAs against con-served genes in C. elegans could induce an avoidance responsein the nematodes.33 The next step, then, was to look for natu-rally occurring RNAs in E. coli with sequence complementarityto C. elegans genes. Liu and colleagues reported on 2 such natu-rally occurring non-coding RNAs in E. coli, OxyS and DsrA,which have sequence complementarity to the C. elegans genesche-2 and F42G9.6, respectively.29 In these experiments, Liuet al. used a variety of behavioral and genetic techniques toassess whether these bacteria which had altered expression ofthese non-coding RNAs had any effect on C. elegans which fedon them. Their paper reports avoidance of E. coli over-express-ing the OxyS ncRNA, as well as a variety of other chemosensorydeficiencies in worms fed on OxyS expressing bacteria. Further,they report increased lifespan in worms fed on DsrA deficientbacteria. However, there is no direct evidence in this paper that

RNA BIOLOGY 417

these physiologic changes are the direct result of ingestedncRNAs, in fact, they show that these effects are still observedin mutants of sid-1 and sid-2, which are required for canonicalenvironmental RNAi. In a follow up to this work, Akay and col-leagues,1 showed that there is no activation of the canonicalRNAi pathway by showing the absence of primary and second-ary siRNAs mapping to the OxyS RNA via small RNAsequencing.

It is also important to note that E. coli is not a significantnatural food source for C. elegans.38 In this paper, Samuel andcolleagues collected likely C. elegans microbiomes from a vari-ety of decaying vegetation in which these worms are oftenfound. These were classified using 16S sequencing and, whileno sequences matching Escherichia were found, many isolatescontained other Enterobacteriaceae in low abundance. Samueland colleagues also measure the time to adulthood of wormsfed on different natural bacteria and found that the differentbacteria can accelerate or delay development. In addition, theyshowed that the effect was not purely nutritional. While activa-tion of pathogenicity responses to toxic bacteria are likely thecause of these effects, it will be interesting to see if environmen-tal RNAi may also play a role.

It may be however, that despite the effectiveness of bacteri-ally produced RNAs to silence C. elegans genes in the labora-tory, that this simply does not occur naturally. It would requirethe co-evolution of the sequences across kingdoms, which,while not unprecedented, requires repeated interaction of thespecies over evolutionary time scales. One alternative may bethat environmental RNAi is a mechanism of communicationbetween worms. One could imagine that feeding on the corpsesof worms from the previous generation could provide informa-tion via RNA which could be interpreted using the RNAimachinery. Interestingly, C. elegans, under stressful conditionssuch as starvation, exhibit a so-called bag-of-worms phenotype,where embryos hatch inside the cuticle of the mother20 Thismay provide one mechanism for the transmission of informa-tion vertically via environmental RNAi. One can speculate thatthis might be useful for relaying very recent information fromthe parental generation.

Initially proposed by Lisa Timmons and Andy Fire, environ-mental RNAi could also function in antiviral immunity.48 Therecent discovery of a first C. elegans virus, the Orsay RNA virus,10 allowed this to be tested. Indeed, C. elegans uses RNAi inantiviral defense 2,10 and delivery of dsRNA against the viralgenome through bacterial feeding immunizes C. elegans againstthe virus.3 However, the generation of an antiviral RNAiresponse seems to be localized to the infected cells, with no evi-dence that it is either systemic or transgenerational.3

Perspectives

RNAi in C. elegans is potent and specific. Using sequence com-plementarity, information about a specific gene to silence isencoded into RNA molecules. A specific pathway exists for thesemolecules to be taken up from the environment and initiate sys-temic gene silencing. This system has a unique property in thatit is a sort of universal translator. Any system capable of generat-ing a dsRNA molecule can use this system as a communicationchannel. Thus, in contrast to a small molecule based system,

which requires the evolution of a signaling pathway to respondto it, RNAi can be used to affect the expression of specific genesfor which no pre-existing pathway yet exists. This system pro-vides a way for information to be passed vertically, from oneorganism to its offspring, and horizontally, from the environ-ment, and thus, in theory, between organisms.40 Furthermore,this provides a mechanism for information acquired from theenvironment of the parent to be transmitted and to influencethe phenotype of the offspring. Physicist Murray Gell-Mannstated, regarding particle physics, that anything not forbidden iscompulsory. Taking this to heart, surely inappropriately, we lookforward to the first conclusive evidence of RNA mediated com-munication in a natural context in C. elegans. The questions tofollow, such as when is RNA used as a signaling medium in con-trast to other modalities, are surely to be of interest not just toRNA biologists, but to biologists in general.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

ORCID

Fabian Braukmann http://orcid.org/0000-0003-2660-450X

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