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RNA Biology

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

Short versus long double-stranded RNA activationof a post-transcriptional gene knockdown pathway

Nir Shpak, Rivka Manor, Lihie Katzir Abilevich, Ortal Mantal, Keshet Shavit,Eliahu D. Aflalo, Debra Toiber & Amir Sagi

To cite this article: Nir Shpak, Rivka Manor, Lihie Katzir Abilevich, Ortal Mantal, KeshetShavit, Eliahu D. Aflalo, Debra Toiber & Amir Sagi (2017): Short versus long double-strandedRNA activation of a post-transcriptional gene knockdown pathway, RNA Biology, DOI:10.1080/15476286.2017.1356567

To link to this article: http://dx.doi.org/10.1080/15476286.2017.1356567

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Accepted author version posted online: 17Aug 2017.Published online: 17 Aug 2017.

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RESEARCH PAPER

Short versus long double-stranded RNA activation of a post-transcriptionalgene knockdown pathway

Nir Shpaka, Rivka Manora, Lihie Katzir Abilevicha, Ortal Mantala, Keshet Shavita, Eliahu D. Aflaloa, Debra Toibera,and Amir Sagia,b

aDepartment of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; bNational Institute for Biotechnology in the Negev, Ben-GurionUniversity of the Negev, Beer-Sheva, Israel

ARTICLE HISTORYReceived 26 April 2017Accepted 11 July 2017

ABSTRACTRNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving theinternalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, completesex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking downthe transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA ofthe entire Mr-IAG ORF sequence (dsMr-IAG – 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragmentsshowing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered theMr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in ourM. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptionalpathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated byobserving Mr-SID1 specific upregulation dependent on dsRNA lengths, while attempted loss-of-functionexperiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths,provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway.The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologiesin aquaculture and environmental applications. Unlike reports of RNAi driven by the administration ofsmall dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo,suggesting further complexity in the context of the entire organism.

KEYWORDSArgonaute; Crustacea; Dicer;dsRNA; Macrobrachiumrosenbergii; M. rosenbergiiinsulin-like androgenichormone (Mr-IAG); SID1;systemic RNAi

Introduction

RNA interference (RNAi) has gained wide acceptance as aneffective tool not only in molecular and gene function research,but also in a variety of technological applications. RNAi isbased on an endogenous cellular defense mechanism againstdouble-stranded RNA (dsRNA) viruses.1 Potent and specificinterference at the post-transcriptional-mRNA level by usingsynthetic dsRNA complementary to endogenous sequences wasfirst observed in Caenorhabditis elegans.2

Gene expression interference through dsRNA is achievablefollowing in-vivo dsRNA administration through feeding,immersion and injection.3,4 Post administration, dsRNA isdelivered into the cytoplasm from the extracellular matrixeither by SID1, a specific dsRNA passive channel,5-7 or throughendocytosis.8 In the cytosol, long dsRNA fragments are proc-essed by Dicer2, a ribonuclease III-related enzyme, into 21–22-bp fragments widely known as siRNAs.9 These siRNAs areincorporated with the Argonaute protein (Ago), the main com-ponent of the multiprotein RNA-induced silencing complex(RISC). In its unwound state, the antisense of the dsRNA facili-tates specific identification of the mRNA targeted for endonu-cleolytic cleavage.10

In general and similar to other non-coding RNAs (ncRNAs),dsRNA molecules are involved in many cellular pathways forregulating eukaryote gene expression during various lifestages.11 These ncRNAs take part in different cellular pathwaysand are all associated with Ago protein family members.12

Interference with gene expression takes place not only throughthe post-transcriptional, siRNA pathway, it can also occur atthe transcriptional level, via the miRNA pathway, in whichsmall ncRNAs are processed into miRNA molecules.13-15 SuchmiRNAs could regulate gene expression in the post-transcrip-tional pathway involving Dicer2 and Ago2. On the other hand,miRNA is also recognized by nuclear proteins, such as Dicer1,resulting in the generation of siRNAs that interact with Ago1,the main component of the RNA-induced initiation transcrip-tional silencing (RITS) complex. These proteins recruit nascentchromatin and chromatin modifying complexes resulting inchromatin remodeling and heterochromatic silencing.16-20 Agofamily proteins are also involved in ncRNAs associated withtransposable element regulation pathways, in which Ago3, aprotein that plays an important role in transcriptional regula-tion, participates.21

CONTACT Amir Sagi sagia@bgu.ac.il Ben Gurion University of teh Negev, P.O.Box 653, Beer Sheva 84105, Israel.Supplemental data for this article can be accessed on the publisher’s website.

© 2017 Taylor & Francis Group, LLC

RNA BIOLOGY2017, VOL. 0, NO. 0, 1–10https://doi.org/10.1080/15476286.2017.1356567

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https://crossmark.crossref.org/dialog/?doi=10.1080/15476286.2017.1356567&domain=pdf&date_stamp=2017-10-11mailto:sagia@bgu.ac.ilhttps://doi.org/10.1080/15476286.2017.1356567https://doi.org/10.1080/15476286.2017.1356567

Using the RNAi method, a biotechnological application wasdeveloped in the aquaculture field for the culture of preferredall-male monosex populations of the giant freshwater prawn,Macrobrachium rosenbergii.22,23 This species exhibits a dimor-phic growth pattern, in which males grow faster and reachlarger final sizes than females at harvest, rendering the cultureof all-male populations economically beneficial.24,25

In crustaceans, sex differentiation is regulated by the andro-genic gland, a male specific endocrine organ. The activity of thegland is mediated in decapod crustaceans through the insulin-like androgenic gland hormone (IAG).26,27 Administration ofdsRNA complementary to the entire open reading frame (ORF)sequence of M. rosenbergii-IAG (Mr-IAG ORF – 518-bp) causesa dramatic knockdown that leads to an approximately 99%decrease in transcript level.22,28 At the phenotypic level, Mr-IAGknockdown causes the cessation of spermatogenesis and theinhibition of secondary masculine sex character development.28

Furthermore, dsMr-IAG injection at a critical juvenile stagecauses a full and functional sex reversal of genetic males (ZZ)into 'neo-females'. When crossed with regular males, such neo-females produce the preferred all-male progeny.22

As in many other eukaryotic species, in several crustaceanspecies the above-mentioned knockdown pathways are highlyconserved and have important roles in gene transcriptional andpost-transcriptional regulation, viral defense and the suppres-sion of transposable elements.29,30 The current study examinedthe expression of the Dicer and Argonaute gene paralogs inM. rosenbergii following dsMr-IAG administration with theaim of elucidating possible Mr-IAG knockdown pathways,whether transcriptional, post-transcriptional or both.

Gene function studies and therapeutic manipulations thatuse dsRNA, which trigger RNAi pathways, typically exploitshort dsRNA, like siRNA or shRNA.4,31,32 These short dsRNAmolecules, however, failed to cause Mr-IAG knockdown, in ourpreliminary work to the current study. Therefore, we sought todetermine the minimum dsRNA length required to obtainMr-IAG knockdown as effective as that achieved by the entireORF sequence (518-bp). Furthermore, we also examined thepossible cellular mechanism behind this manipulation andthe changes that occurred in the expression levels of Mr-IAGand several RNAi associated genes, such as the SID1 and Dicerparalogs, in response to different dsMr-IAG lengths.

Results

Effect of dsRNA fragment lengths shorter than the full ORFonMr-IAG knockdown efficiency

Attempts to knock down Mr-IAG expression levels usingshorter dsMr-IAG fragments showed a clear correlationbetween dsRNA fragment length and Mr-IAG knockdownefficiency, i.e., the longer the dsRNA fragment, the greater theknockdown of Mr-IAG expression levels (Fig. 1). Of the fourlengths tested, only the 250-bp dsRNA fragment caused a sig-nificant knockdown effect similar to the entire ORF (»99%decrease compared with the negative control, F(2,15) D 18.871,P-value < 0.0001). The relative Mr-IAG expression levels whileusing the 250-bp fragments were significantly different fromthat of the negative control and strikingly similar to the effectsof the entire ORF sequence (P-value < 0.001 and P-value D

0.93, respectively). A pair of dsRNA fragments of 100-bp (Serialno. 100_2 and 100_5, Fig. S1A) caused »75% decrease inexpression, which was significantly different from expressionlevels in both the negative control group and the entire ORFgroup (P-value D 0.034 and P-value < 0.001, respectively).Attempts to improve the knockdown efficiency of the 100-bpfragments by covering the entire ORF sequence with a mixtureof 100-bp fragments did not yield better results (See Fig. S2). Asimilar mixture of 75-bp dsRNA fragments caused a minordecrease in Mr-IAG transcript level, but statistically, that resultwas similar to those of both the negative control and the entireORF injected groups (P-value D 0.37 and 0.178, respectively).Transcription level in the control (non-injected) group was sig-nificantly different from that of the entire ORF injected group(P-value D 0.012). All attempts to knockdown Mr-IAGexpression using a mixture of 19-bp, short interference RNA(siRNA) did not cause down regulation compared with the neg-ative control group (P-value D 1.0) and were significantly dif-ferent from the results for the entire ORF (P-value < 0.01). Inall the distinct length dependency experiments (mixture of 19-bp, 75-bp, 100-bp or 250-bp fragments), Mr-IAG expressionlevels in the negative control groups and in the entire ORFinjected groups were significantly different.

Presence and phylogeny of knockdown-pathway-relatedgenes inM. rosenbergii

To understand the difference in M. rosenbergii in the acti-vation of RNAi and its dependence on dsRNA length com-pared with its activation in other RNAi pathways, weexamined the main RNAi associated genes, members of theDicer and Argonaute families. Mining our M. rosenbergiitranscriptomic library resulted in multiple sequence align-ments with the Mr-Dicer family based on putative aminoacid sequences showing cluster deviations for both Dicer1

Figure 1. dsRNA length knockdown dependency. (A) Effect of administered dsMr-IAG lengths (19-bp, 75-bp, 100-bp, and 250-bp) on Mr-IAG transcript levels in 4 dis-tinct in-vivo experiments. Negative control – non-injected group, Positive control –entire ORF injected group (518-bp). (B) A general transcript reduction patternfollowing injection of dsMr-IAG at different lengths.

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and Dicer2 (Fig. 2A). M. rosenbergii Dicer1 (Mr-Dicer1)showed homologies to Marsupenaeus japonicus Dicer1,Penaeus monodon Dicer1, Litopenaeus vannamei Dicer1 andDrosophila melanogaster Dicer1 (83%, 71%, 70% and 67%,respectively). Mr-Dicer2 showed homologies to Lv-Dicer2,Pm-Dicer2 and Dm-Dicer2 (51%, 51% and 27%, respec-tively). Homology of 32% was found between the Mr-Dicerparalogs.

Phylogenetic analysis based on multiple amino acidsequence alignments of 3 Ago candidates demonstrated devia-tions into only two clusters: cluster A for Ago2 and Ago3, andcluster B for Ago1 (Fig. 2B). Mr-Ago1 showed significant

homology to Ago1 orthologs from other crustacean speciessuch as Panulirus interruptus Ago1, Mj-Ago1 and Lv-Ago1(99%, 98% and 98%, respectively) and to Dm-Ago1 (86%homology). The cases of Argonaute2 and Argonaute3 were dif-ficult to differentiate by phylogenetic analysis. Homologies of43% and 38% were noted between Mr-Argonaute paralogs ofMr-Ago1 to Mr-Ago2 and Mr-Ago3, respectively. A higher,homology (48%) was found between Mr-Ago2 and Mr-Ago3.

To elucidate which knockdown pathway was activated as aresponse to dsMr-IAGmanipulation, dsMr-IAG was administeredand RNAi associated gene induction (Dicer and Ago families) wastested. Mr-IAG expression levels were significantly lower, »99%of control (t(10) D 18.608, p-value< 0.0001), as a result of dsMr-IAG injection (Fig. 3A). Comparing the expression levels of genesrelated to the RNAi mechanism between the group injected withdsMr-IAG to a non-injected group demonstrated that Mr-Dicer2was significantly upregulated in the injected group (t(6) D¡4.873, p-value< 0.01) (Fig. 2B) whileMr-Dicer1 expression lev-els were similar to those in the non-injected group (p-Value D0.128). Similarly, in the Ago gene family, only one candidate,Mr-Ago2, was upregulated in response to dsMr-IAG injection(p-value < 0.001) (Fig. 3B), while Mr-Ago1 and Mr-Ago3expression levels were similar to those in the non-injected group(t-test, t(13) D ¡1.091, P-value D 0.295 and Mann-WhitneyU-test, P-value D 0.779, respectively).

Mr-SID1, knockdown pathway and dsRNA length

Mr-SID1, which encodes for a passive channel with specificityto dsRNA molecules, was found by comparing the L. vannameiSID1 amino acid sequence to our M. rosenbergii transcriptomiclibrary, resulting in a transcript with 52% homology. This tran-script (Mr-SID1) was fully sequenced, and 11 transmembraneregions were predicted in its putative protein sequence by abioinformatics hydrophobicity evaluation (Fig. S3A). Phyloge-netic analysis demonstrated that SID1 is conserved betweendifferent arthropod species (Fig. S3B).

With the aim of confirming the function of Mr-SID1, itsexpression levels were measured in response to administrationof dsRNA and DNA (both of GFP sequence) and PBS as a neg-ative control (Fig. 4A). Mr-SID1 was upregulated only in thepresence of dsRNA (F(2,19) D 7.19, p-value D 0.004), showingthe specificity of Mr-SID1 to dsRNA. Furthermore, loss offunction of Mr-SID1 showed a lethal effect after two consecu-tive injections of dsMr-SID1 and dsGFP (Fig. 4B). In all thecontrol groups, including PBS, dsGFP and dual injections oftwo exogenous dsRNA (dsGFP C dsRB), high survival rateswere demonstrated (100%, 100% and 87%, respectively). Allthe dsMr-SID1 and dsGFP injected individuals died during the24 h following the second injection.

To conclude the study, expression levels of Mr-SID1 andthe other components of the RNAi mechanism were tested inresponse to different fragment lengths of dsMr-IAG based onthe same length dependency experiments shown in Fig. 1.The correlation clearly showed that dsMr-IAG fragmentlength was positively correlated with Mr-SID1 upregulation(Fig. 5A). A significant induction of Mr-SID1 expression lev-els was measured by dsMr-IAG fragment lengths of 100-bp(P-value D 0.01) and 250-bp (P-value D 0.002), and this

Figure 2. Phylogenetic analysis knockdown pathway associated genes. Phyloge-netic analysis based on multiple alignments using Mega 6.0 software. The neigh-bor-joining tree was constructed according to multiple alignments of the putativeamino acid sequences: (A) Dicer constructed according to phylogenetic analysis offull amino acid sequences. (B) Argonaute phylogenetic analysis constructedaccording to multiple alignments of PIWI domain amino acid sequences. Tree scaleindicates the evolutionary distance by tree branch lengths, which are proportionalto sequence differences. Bootstrap values represented by numbers next to thebranches. GenBank accession numbers are in Table S5.

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activity was similar to the upregulation of Mr-SID1 expressionlevels following injections of the entire ORF (P-value D0.003 and 0.027, respectively). The administration of a mix-ture of 19-bp fragments did not cause Mr-SID1 induction,whose expression levels were similar to that of the negativecontrol. The expression levels of Mr-SID1 following the injec-tion of 75-bp fragments of dsMr-IAG were different fromthose of both the negative control and the entire ORF(P-value < 0.001, for both).

The same correlation shown above with respect to Mr-SID1and different dsMr-IAG fragment lengths was also found forMr-Dicer2 with the exception of the induction caused by 100-bpdsRNA fragments (Fig. 5B). The expression levels of Mr-Dicer2in response to 100-bp fragments of dsRNA were significantlyhigher than the entire ORF injected (P-value D 0.041), andthose of both the 100-bp fragment and the entire ORF were sig-nificantly higher than that of the non-injected negative controlgroup (P-value < 0.001, both). Administrations of 75-bp and250-bp fragments of dsRNA elicited higher Mr-Dicer2 expressionlevels than those of the non-injected group (P-value D 0.005and P-value < 0.001, respectively) and, in contrast to the resultsobtained for the 100-bp fragments, showed similar Mr-Dicer2expression levels to that obtained in response to injection of theentire ORF (p-value D 0.7 and 0.98, respectively). In responseto administration of the mixture of 19-bp fragments, the expres-sion levels of Mr-Dicer2, similar to those of Mr-SID1, were notaffected, and its expression levels were similar to those in thenon-injected group (p-value D 0.99).

As a reference, we used the expression levels of Mr-Dicer1from the miRNA pathway. This gene remained at the same lowlevels in all the treatments, thus demonstrating the absence of acorrelation with dsMr-IAG fragment length (Fig. 5C).

Discussion

TheMr-IAG knockdown pathway

Full and functional sex reversal of male crustaceans intofemales caused by the use of RNAi demonstrates the vastpotential of RNAi-based biotechnologies in aquaculture and

environmental applications.3,33,34 The use of such a biotech-nology was previously suggested to be safe based on thetemporal nature of the intervention and its lack of anyovertly apparent long-term consequences.23,35 These studiespresented evidence based on social structure, growth perfor-mance and masculine reproductive characters at the ana-tomic and molecular levels. Moreover, our observations thatthe elapsed times until disappearance of the exogenousdsRNA from prawn tissues and the return of Mr-IAG tran-script levels to normal during the post silencing periodwere comparable, suggesting that RNAi-based technology istemporal and indeed safe.23,35 The current study, whichexamined the mechanism of the knockdown pathway, pro-vided additional support for the temporal nature of theRNAi-based production of all-male cultures and rejects thepossible involvement of permanent or epigenetic interfer-ence processes on the genome associated with the transcrip-tional silencing pathway.

Three known RNAi pathways, i.e., micro RNAs (miRNA),short interfering RNA (siRNA) and PIWI interacting RNA(piRNA), are associated with Argonaute protein familymembers.12 In the current study, the Dicer and Ago homo-logs were discovered and fully sequenced in M. rosenbergii.The clustering of each homolog to other members fromrelated species further supports the correct identification ofthe distinct Mr-Dicer and Mr-Ago members allowing theexamination of the response of each homolog transcript levelto dsMr-IAG administration. Only transcripts of Mr-Dicer2and Mr-Ago2, essential components for RNAi response andpost-transcriptional silencing, were significantly increasedafter dsMr-IAG injection. Other transcripts in these families,such as Mr-Ago1 and Mr-Dicer1 associated with nucleartranscriptional silencing16,17 or Ago3 associated withpiRNA,21 were not affected by the introduction of dsMr-IAG. Since the Dicer2 and Ago2 proteins are known to beinvolved in the post-transcriptional pathway where theyexert temporal cytoplasmic effects,12,36 the results of thepresent study support both the temporal nature of the effectsof dsMr-IAG administration and the safety of its use inaquaculture and environmental applications.

Figure 3. Expression levels of RNAi associated genes in response to entire ORF dsMr-IAG injection. (A) Mr-IAG expression level in dsMr-IAG injected group (entire ORF) vs.non-injected group (Control). (B) Expression levels of Mr-Dicer and Mr-Argonaute paralogs in the same experimental groups as in (A).

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Post-transcriptional knockdown pathway and dsRNAlength

The current study also focused on the ability to knock downMr-IAG using fragments with lengths shorter than the entireORF. Gene knockdown in various organisms was demonstratedusing short dsRNA fragments (siRNA) for loss of function pro-cedures, mostly in-vitro.37-39 Thus, we attempted to knockdown Mr-IAG in-vivo using siRNA. These attempts failed, elic-iting the need to examine the correlation between dsRNAlength and knockdown success. A clear relationship betweendsMr-IAG lengths and Mr-IAG transcript levels was found, inwhich the longer the administered dsMr-IAG fragment, the

lower the Mr-IAG transcript levels. A similar tendency wasdemonstrated in D. melanogaster.6 To gain insight into thisphenomenon, different dsMr-IAG fragment lengths were evalu-ated for their effects on the expression levels of genes associatedwith the post-transcriptional knockdown pathway.

To investigate the mechanism of action, Mr-SID1, a passivetransmembrane channel that specifically delivers dsRNA intosomatic cells,5,7 was sequenced and aligned to orthologs fromarthropod species. Mr-SID1 is the main gate for passive dsRNAuptake into the cell and indeed its transcript levels were onlyupregulated in the presence of dsRNA. The results are sup-ported by those of Shih and Hunter,7 who examined the differ-ences in membrane conductance in SID1-expressing cells inthe presence of dsRNA and DNA and showed that conductancechanged only in the presence of dsRNA. Based on the aminoacid homology of Mr-SID1 to other species with the same pre-dicted 11 transmembrane regions and based on the results ofthe functional test of Mr-SID1, we suggest that Mr-SID1 is thedsRNA-gated channel inM. rosenbergii.

Yet SID1 is not the only way known for dsRNA delivery intothe cytosol. Many metazoan cells can deliver an exogenousdsRNA via receptor mediated endocytosis, an uptake mecha-nism that seems to be evolutionarily conserved.8 To determinewhether Mr-SID1 is the main avenue for the delivery of dsRNAinto M. rosenbergii cells, we attempted to knock down SID1,after which we planned to try SID1 knockdown in parallel withdsMr-IAG administration. The latter experiment, however,could not be performed since the effect of dsMr-SID1 adminis-tration was lethal unlike the cases of other documented RNAi-associated genes.40,41 It should be noted that dsMr-SID1 wasdesigned based on a wide M. rosenbergii transcriptomiclibrary42 and found to be specific to the Mr-SID1 sequence,thereby minimizing the probability of off-target effects. How-ever, due to the lack of a full genome sequence in our studyorganism, lethality caused by off-target effects could not becompletely ruled out. In addition, while we also cannot rule outthe possibility that dsRNA was internalized through endocyto-sis, we suggest that SID1 is a major component in dsRNA traf-ficking. This is supported by results from SID1 non expressingD. melanogaster S2 cells, in which dsRNA is endocytoticallydelivered,8 while the expression of SID1 in those cells increasesthe efficiency of gene knockdown.6 In the M. rosenbergii sys-tem, the presence and differential activity of Mr-SID1 seem tosuggest that it plays a crucial role in regulation of systemicRNAi and the cell’s defense mechanism against viruses.

The present study showed a positive correlation betweeninjected dsMr-IAG fragment length and Mr-SID1 transcriptinduction level. In contrast to our findings, another study thatexamined C. elegans SID1 expression in D. melanogaster S2cells did not find it to be selective to dsRNA length.43 Theauthors of the latter article suggested that dsRNA length depen-dency probably does not occur through SID1.43 It is thussuggested that the tendency of Mr-SID1 transcripts to respondto dsRNA lengths is regulated by factors downstream, and notupstream, of the dsRNA delivery, and that it is not self-regu-lated by the transmembrane channel itself. It is also suggestedthat dsRNA delivery depends on the dsRNA retention ability ofthe Dicer-RISC complex. Insofar as SID1 is a passive channel,dsRNA retention by Dicer-RISC is essential to allow the

Figure 4. Mr-SID1 expression in response to dsRNA injection and its loss of func-tion effects. (A) dsRNA specificity demonstrated by a comparison between Mr-SID1expression level in dsRNA-injected compared with dsDNA-injected groups(dsRNA and dsDNA contain the same GFP exogenous sequence) and negative con-trol, PBS-injected group (n D 7 in each group). (B) Survival rates of juvenileprawns following dsMr-SID1 injection (with dsGFP). Controls included the PBS-injected group administered a double volume similar to the volume used in thedual injection of 2 dsRNAs, the dsGFP injected group, and the dsGFP and dsRBinjected group.

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intra-cellular dsRNA concentration to decrease, resulting inmore dsRNA molecules passing into the cell through SID1according to intra- and extra-cellular dsRNA concentrations.43

Mr-Dicer2 transcript levels also changed in response todsMr-IAG length but without a clear, linear tendency as wasfound for Mr-SID1. In the case of Mr-Dicer2, the highest upre-gulation was obtained with dsRNA fragments of 100-bp com-pared with the other fragment sizes. Moreover, from amongthe fragment lengths tested, only the 19-bp fragments failed tocause any Mr-Dicer2 upregulation. These results seem to con-tradict observations of knockdown success, which was betterfor fragment lengths of 250-bp and 500-bp. It is suggested thatMr-Dicer2 is influenced by the ability of SID1 to deliver the

dsRNA molecules and that the level of induction depends onthe number of molecules. Among the solutions of the differentfragments at the same dsRNA concentration (in this study5 mg/g body weight), that with the 100-bp fragments containsmany more molecules, and this could explain the finding thatthe highest upregulation obtained here was forMr-Dicer2.

Concluding remarks

For the first time, the Dicer and Argonaute genes were fullysequenced in M. rosenbergii, which enabled us to elucidate theknockdown pathway of Mr-IAG, the target of the manipula-tion that causes the full and functional sex reversal of

Figure 5. RNAi-associated gene expression levels following injection of Mr-IAG dsRNA of different lengths (19-bp, 75-bp, 100-bp, 250-bp, and entire ORF – 518-bp). Mr-SID(A), Mr-Dicer2 (B) and a reference gene, Mr-Dicer1, from the miRNA pathway (C). Expression levels were tested in the RNA extracted from the length dependency experi-ments (Fig. 1).

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M. rosenbergii males into females. This study showed that thedsMr-IAG-based manipulation manifests through a temporal,post-transcriptional pathway, the well-known siRNA pathway.The temporal nature of the latter pathway further supportsthe safety of using such RNAi-based biotechnologies in aqua-culture and environmental applications.

In this study, RNAi-based manipulation of the crustaceaninsulin-like androgenic hormone also demonstrated a cleardependency on dsRNA molecule length, such that dsMr-IAGfragment length was negatively correlated with Mr-IAG tran-script level. Moreover, the induction of genes associated with thepost-transcriptional pathway and SID1 was also differentiallyinfluenced according to dsMr-IAG fragment length. Unlikenumerous clear cases of RNAi driven by the administration ofsmall dsRNA fragments in-vitro, the case presented here demon-strates length dependency when the experiments are performedin-vivo. Our results thus support the need for further study ofRNAi phenomena in the context of the entire organism.

Materials and methods

Animals

M. rosenbergii prawns of the BGU-line were maintained atBen-Gurion University of the Negev (BGU) facilities asdescribed by Shpak et al.44 The all-male progenies originatingfrom crosses between males and 'neo-females’ were supplied bythe Tiran Shipping group through its subcontractor hatchery,Colors Ltd., Hatzeva, Israel. This hatchery holds only geneticmales form the BGU line, and genetic females have never beenallowed at this farm.

All dsRNA length dependency experiments were performedusing small males whose morphotype was determined accord-ing to Kuris et al.45 It should be noted that small males aresexually active and have an active androgenic gland.46

Mr-SID1 loss of function and specific induction of Mr-SID1by dsRNA experiments were performed using post larvae all-male progeny from the all-male hatchery mentioned above.

Synthesis of double-stranded RNA

Eight dsRNA fragments of 19-bp and two fragments of 75-bpfrom the Mr-IAG mRNA sequence were synthesized andsupplied by BioSpring, Frankfurt, Germany (Fig. S1A). FivedsRNA fragments of 100-bp covering approximately the entireMr-IAG ORF and one fragment of 250-bp were synthesized in-vitro in our laboratory (Fig. S1A). A pGEM�-T Easy plasmidcontaining the Mr-IAG open reading frame sequence served asthe template for dsMr-IAG synthesis. The template was ampli-fied by PCR, primed by two gene-specific primers with a T7promotor site at the 5' of one primer (T7P) (see primers andT7 promotor sequences for dsRNA synthesis in Table S1).Primer pairs were as follows: the sense strand was synthesizedusing primer T7P forward vs. reverse primer, while the anti-sense strand was synthesized by T7P reverse vs. forwardprimer. PCR amplicons were electrophoresed on a 1.3% agarosegel and visualized with ethidium bromide and UV light, excisedfrom the gel, and purified with a Accuprep� PCR purificationKit (BIONEER Co., Daejeon, South Korea).

The TranscriptAid T7 High Yield Transcription Kit(Thermo Scientific, Lithuania) was used to generate single-stranded RNA according to the manufacturer’s instructions.RNA molecules were purified by phenol-chloroform (1:1) andammonium-acetate and precipitated with ethanol. Sense andantisense strands were hybridized by incubation at 70�C for15 min, 65�C for 15 min and at RT for 30 min. dsRNA qualitywas assessed on an agarose gel and diluted to 5 mg/ml. dsRNAwas kept at ¡80�C until used.

Mr-IAG knockdown attempts using fragments shorter thanthe entire ORF

Four distinct experiments were performed, one for each dsRNAlength (19-bp, 75-bp, 100-bp, and 250-bp). Each experimentincluded a non-injected group as a negative control and anentire dsMr-IAG ORF injected group as a positive control. It isimportant to note that the determination of which animalswould constitute the non-injected control groups was based onprevious studies, which also used exogenous dsRNA or DDWinjection as described in Lezer et al.35 and in Sharabi et al.,42

with similar results. Each experimental group included 6–8small M. rosenbergii males (Mean body weight D 12.5 g). Sin-gle injections of the different dsMr-IAG lengths (5mg dsRNA/gr body weight) were performed once, and then 48 hours post-injection, prawns were anesthetized in ice cold water, dissectedand total RNA was extracted from their androgenic glandsusing the EZ-RNA Total RNA Isolation Kit (Biological Indus-tries, Beit Haemek, Israel) according to the manufacturer’sinstructions. cDNA was prepared in a reverse-transcriptasereaction containing 1 mg total RNA using a qScript cDNA syn-thesis kit (Quanta Biosciences, Gaithersburg, MD, USA)according to the manufacturer’s instructions. Relative quantifi-cation of Mr-IAG expression levels was obtained as describedin Ventura et al.22 with the FastStart Universal Probe Master(Rox; Roche Diagnostics GmbH) and Universal ProbeLibraryProbe 144 (Roche). Mr-18S (GenBank accession no.GQ131934) was used as a normalizing gene and quantifiedusing specific primers as described in Ventura et al.22 with theabove-mentioned mix and Universal ProbeLibrary Probe 152(Roche). Reactions were performed with the ABI Prism 7300Sequence Detection System (Applied Biosystems).

RNAi associated gene mining

RNAi associated-genes were mined from our compositeM. rosenbergii transcriptomic library, which contains morethan 290 million reads assembled into 108212 contigs, fullydescribed by Ventura, et al.47 and Sharabi, et al.42 The miningwas done based on orthologs from related crustacean speciesusing a BLAST search to reveal Dicer and Ago homologs andSID1sequences. M. rosenbergii-Dicer1 (Mr-Dicer1) was com-pared with P. monodon Dicer1 (GenBank accession numberABR14013.1) and Mr-Dicer2 was compared with L. vannameiDicer2 (GenBank accession number ACF96960.1). Mr-Argo-naute1 was compared with M. japonicus Argonaute1 (GenBankaccession number ADB44074.1). Mr-Argonaute2 was com-pared with M. japonicus Argonaute2 (GenBank accession num-ber AB665954.1). Mr-Argonaute3 was compared with P.

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monodon Argonaute3 (GenBank accession numberAGC95229.1). M. rosenbergii-SID1 was compared L. vanna-mei-SID1 (GenBank accession number HM234688.1).

Putative sequences obtained from the library were computa-tionally translated using the ExPASy Proteomics Server (http://ca.expasy.org/tools/dna.html), and the longest open readingframe was selected. Transmembrane regions of Mr-SID1 wereevaluated using the Simple Modular Architecture ResearchTool (SMART) (http://smart.embl-heidelberg.de/).48

In-vitro validation and sequencing

Total RNA was extracted from the hepatopancreas of dsMr-IAG-injected [5mg/ml] animals, while anesthetized in ice-coldwater, 48 h post-injection. Total RNA was isolated and cDNAwas prepared as described above.

PutativeMr-Dicers (Mr-Dicer1,2), Mr-Agos (Mr-Ago1,2,3) andMr-SID1 sequences were in-vitro validated by PCR using genespecific primers (Table S2). The PCR products were cloned andsequenced as described by Ventura, et al.28 Mr-Dicer2 and Mr-SID1 5' end were obtained by 5' rapid amplification of cDNAends (RACE 5') performed with the Clontech SMART RACE kit(BD Biosciences, Palo Alto, CA) following the manufacturersprotocol. PCR was performed using a gene-specific reverseprimer (Table S3) and the Universal Primers Mix (UPM) pro-vided in the kit. To confirm the obtained sequence, 5' RACEproduct was amplified by PCR using a reverse nested primercloser to the 5' end (Table S3) and a UPM nested primer, alsoprovided in the kit. RACE 5' and nested 5' products were clonedand sequenced as described above. To ensure the quality of thesequence, each region of the gene was sequenced four times.

The 3' end of Mr-Ago3 was obtained by 3' rapid amplifica-tion of cDNA ends (RACE) as described above using a gene-specific forward primer (Table S3) and a UPM provided in thekit, and the PCR products were cloned and sequenced. To con-firm the obtained sequence, the 3' RACE product was amplifiedby PCR using a forward nested primer closer to the 3' end(Table S3). This product was cloned and sequenced asdescribed above. To ensure the quality of the sequence, eachregion of the gene was sequenced four times.

Phylogenetic analysis

Known sequences of Dicer and Ago from related Crustaceanspecies and SID1 sequences from different species wereobtained from the GenBank database. A phylogenetic neigh-bor-joining tree was constructed using Mega 6.0 software(Molecular Evolutionary Genetics Analysis, version 6.0)according to ClustalW multiple sequence alignments of theputative amino acid sequences of Dicer, Argonaute and SID1.Bootstrap values (%) of 1000 replicates were calculated for eachnode of the consensus tree attained.49

Mr-IAG knockdown pathway determination

To elucidate the Mr-IAG knockdown pathway, 15 smallM. rosenbergii males (mean body weight »15 gr) were dividedinto two groups, the non-injected group (n D 8) and theinjected group (n D 7), whose members were injected with

the entire dsMr-IAG ORF [5mg dsRNA/gr body weight] by amicro injector. Androgenic glands and hepatopancreases weredissected for total RNA extraction, 48 h post injection asdescribed above. Total RNA was isolated and cDNA was pre-pared as described above. Mr-IAG expression levels weredetected from total RNA extracted from androgenic glands toverify Mr-IAG knockdown success. Relative quantification ofMr-IAG expression levels was obtained as described above.

Relative quantification of Mr-Dicer (Mr-Dicer1,2) andMr-Argonaute (Mr-Ago1,2,3) expression levels were testedusing the total RNA isolated from the hepatopancreas,preliminarily showing high expression levels. Mr-Dicers andMr-Argonautes expression levels were obtained using forwardand reverse primers (Table S4) with FastStart Universal ProbeMaster (Rox; Roche Diagnostics GmbH) and UniversalProbeLibrary Probes (Roche) (Table S4). For all these relativequantifications, Mr-18S was also quantified by real-time RTPCR as a normalizing gene as described above.

Double-strand RNA andMr-SID1 induction

To confirm the role of SID1 in M. rosenbergii, juvenile males(n D 22) (mean weight D 0.22 gr) were divided into threegroups: phosphate-buffered saline (PBSX1) injected (n D 8),DNA-GFP injected (n D 7) and dsRNA-GFP injected (n D7). dsRNA-GFP was synthesized as described above. DNA frag-ments that contained the same GFP sequence were amplifiedby PCR. PCR products were purified using NucleoSpin� Geland PCR Clean-up (MACHEREY-NAGEL GmbH & Co.,D€uren, Germany). The procedure included two injections of5mg per g body weight each every 24 h. Prawns were anesthe-tized in ice cold water and their hepatopancreases were dis-sected 24h post second injection. Total RNA was extracted andcDNA was prepared as described above. Relative quantificationof Mr-SID1 expression levels was performed as described above(for primers and probe, see Table S4).

The effect ofMr-SID1 knockdown

With the aim of examining the crucial role of Mr-SID1 togene knockdown in M. rosenbergii, the effect of Mr-SID1knockdown was studied. Forty juvenile males (mean bodyweight D 0.67 gr) were divided to four groups: DDWinjected, dsGFP injected, dual injection group of dsRB anddsGFP (two exogenous dsRNA) and dual injection group ofdsGFP and dsMr-SID1 (the purpose of dsGFP was to induceMr-SID1 expression level). The procedure included twoinjections of 10mg mixed dsRNA per g body weight with24 h interval and survival rates were monitored. Synthesisof dsGFP was done as described above. dsRB, an exogenousgene, was synthesized as described by Lezer et al.35 Thesynthesis of dsMr-SID1 was done as described above usingspecific primers (Table S1)

RNAi-associated genes expression levels as a responseto different dsMr-IAG length

RNAi-associated gene expression levels (Mr-SID1, Mr-Dicer2and Mr-Dicer1) were measured using the same RNA isolated

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http://ca.expasy.org/tools/dna.htmlhttp://ca.expasy.org/tools/dna.htmlhttp://smart.embl-heidelberg.de/

from the prawns that was used in the Mr-IAG knockdownattempts, with the shorter dsMr-IAG fragments describedabove including control groups. Mr-SID1, Mr-Dicer2 andMr-Dicer1 were quantified by real-time RT-PCR using theabove described primers and Universal ProbeLibrary Probes ve.

Statistical analysis

Final real-time RT-PCR relative quantification values (RQ) werecalculated using the formula 2¡ddCt (built-in feature in the ABIPrism 7300 Sequence Detection System, Applied Biosystems) foranalyzing qPCR results. The RQ values in the different experi-ments were analyzed by Statistica v12.0 software (StatSoft, Ltd.,Tulsa, OK, USA). For all multiple comparisons, the tests wereselected according to assumptions of homogeneity of the varian-ces through Levene’s test and normal distribution of the residualsas accepted. For all comparisons between two groups, the testswere selected according to the normal distribution of the samplesand the homogeneity of the variances through a P-variance test.

The results of knockdown pathway elucidation experimentswere analyzed using a t-test for independent variables forMr-IAG, Mr-Dicer2 and Mr-Ago1 and the Mann-WhitneyU-test for the results of Mr-Dicer1, Mr-Ago2 and Mr-Ago3. Theresults of evaluations ofMr-IAG response to shorter dsRNA frag-ment lengths were analyzed using One-way ANOVA after log(X)transformation and Post-hoc Tukey HSD for 100-bp and 250-bpexperiments, One-Way ANOVA using welch correction andTukey HSD for unequal samples for the 75-bp experiment, andKruskal-Wallis was used for the 19-bp mixture experiment. Thedifferences in Mr-SID1 expression levels were analyzed usingOne-way ANOVA after log(XC1) transformation and Post-hocTukey HSD. The correlation between dsRNA lengths and Mr-SID1 expression were analyzed using the Kruskal-Wallis test forthe 19-bp, 100-bp and 250-bp experiments. One-Way ANOVAusing welch correction and Tukey HSD for unequal sampleswere used to examine the correlation between 75-bp fragmentsand the Mr-SID1 transcript. The correlation between dsRNAlength and Mr-Dicer2 were analyzed using One-way ANOVAafter log(XC1) transformation and Post-hoc Tukey HSD.

Acknowledgments

This study was supported in part by the United States–Israel BinationalAgricultural Research and Development Fund (BARD, Grant No. IS-4493–12), the Israel Science Foundation (ISF) within the ISF-UGC jointresearch program framework (grant No. 2728/16) and the Israel ScienceFoundation and the National Natural Science Foundation of China Grant605/14. All-male progeny were supplied by Tiran Shipping, through theirsubcontractor Colors Ltd., Hatzeva, Israel.

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AbstractIntroductionResultsEffect of dsRNA fragment lengths shorter than the full ORF on Mr-IAG knockdown efficiencyPresence and phylogeny of knockdown-pathway-related genes in M. rosenbergiiMr-SID1, knockdown pathway and dsRNA length

DiscussionThe Mr-IAG knockdown pathwayPost-transcriptional knockdown pathway and dsRNA lengthConcluding remarks

Materials and methodsAnimalsSynthesis of double-stranded RNAMr-IAG knockdown attempts using fragments shorter than the entire ORFRNAi associated gene miningIn-vitro validation and sequencingPhylogenetic analysisMr-IAG knockdown pathway determinationDouble-strand RNA and Mr-SID1 inductionThe effect of Mr-SID1 knockdownRNAi-associated genes expression levels as a response to different dsMr-IAG lengthStatistical analysis

AcknowledgmentsReferences