Short Versus Long Double-Stranded RNA Activation of a Post-Transcriptional Gene Knockdown Pathway

RNA Biology

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Short versus long double-stranded RNA activation of 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, Keshet Shavit, Eliahu D. Aflalo, Debra Toiber & Amir Sagi (2017): Short versus long double-stranded RNA 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: 17 Aug 2017. Published online: 17 Aug 2017.

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Download by: [Ben Gurion University of the Negev] Date: 07 November 2017, At: 01:26 RNA BIOLOGY 2017, VOL. 0, NO. 0, 1–10 https://doi.org/10.1080/15476286.2017.1356567

RESEARCH PAPER Short versus long double-stranded RNA activation of a post-transcriptional gene 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-Gurion University of the Negev, Beer-Sheva, Israel

ABSTRACT ARTICLE HISTORY RNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the Received 26 April 2017 internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete Accepted 11 July 2017 sex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking down KEYWORDS the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of Argonaute; Crustacea; Dicer; the entire Mr-IAG ORF sequence (dsMr-IAG – 518bp). Interestingly, in-vivo knockdown success and dsMr- dsRNA; Macrobrachium IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments rosenbergii; M. rosenbergii showing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered the insulin-like androgenic Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our hormone (Mr-IAG); SID1; M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional systemic RNAi pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 speciﬁc upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments 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 biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

Introduction RNA interference (RNAi) has gained wide acceptance as an In general and similar to other non-coding RNAs (ncRNAs), effective tool not only in molecular and gene function research, dsRNA molecules are involved in many cellular pathways for but also in a variety of technological applications. RNAi is regulating eukaryote gene expression during various life based on an endogenous cellular defense mechanism against stages.11 These ncRNAs take part in different cellular pathways double-stranded RNA (dsRNA) viruses.1 Potent and speciﬁc and are all associated with Ago protein family members.12 interference at the post-transcriptional-mRNA level by using Interference with gene expression takes place not only through synthetic dsRNA complementary to endogenous sequences was the post-transcriptional, siRNA pathway, it can also occur at ﬁrst observed in Caenorhabditis elegans.2 the transcriptional level, via the miRNA pathway, in which 13-15

Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 Gene expression interference through dsRNA is achievable small ncRNAs are processed into miRNA molecules. Such following in-vivo dsRNA administration through feeding, miRNAs could regulate gene expression in the post-transcrip- immersion and injection.3,4 Post administration, dsRNA is tional pathway involving Dicer2 and Ago2. On the other hand, delivered into the cytoplasm from the extracellular matrix miRNA is also recognized by nuclear proteins, such as Dicer1, either by SID1, a specific dsRNA passive channel,5-7 or through resulting in the generation of siRNAs that interact with Ago1, endocytosis.8 In the cytosol, long dsRNA fragments are proc- the main component of the RNA-induced initiation transcrip- essed by Dicer2, a ribonuclease III-related enzyme, into 21–22- tional silencing (RITS) complex. These proteins recruit nascent bp fragments widely known as siRNAs.9 These siRNAs are chromatin and chromatin modifying complexes resulting in incorporated with the Argonaute protein (Ago), the main com- chromatin remodeling and heterochromatic silencing.16-20 Ago ponent of the multiprotein RNA-induced silencing complex family proteins are also involved in ncRNAs associated with (RISC). In its unwound state, the antisense of the dsRNA facili- transposable element regulation pathways, in which Ago3, a tates specific identification of the mRNA targeted for endonu- protein that plays an important role in transcriptional regula- cleolytic cleavage.10 tion, participates.21

CONTACT Amir Sagi [email protected] 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 2 N. SHPAK ET AL.

Using the RNAi method, a biotechnological application was developed in the aquaculture field for the culture of preferred all-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 reach larger final sizes than females at harvest, rendering the culture of all-male populations economically beneficial.24,25 In crustaceans, sex differentiation is regulated by the androgenic gland, a male specific endocrine organ. The activity of the gland is mediated in decapod crustaceans through the insulin- like androgenic gland hormone (IAG).26,27 Administration of dsRNA complementary to the entire open reading frame (ORF) sequence of M. rosenbergii-IAG (Mr-IAG ORF – 518-bp) causes a dramatic knockdown that leads to an approximately 99% decrease in transcript level.22,28 At the phenotypic level, Mr-IAG knockdown causes the cessation of spermatogenesis and the inhibition of secondary masculine sex character development.28 Furthermore, dsMr-IAG injection at a critical juvenile stage causes a full and functional sex reversal of genetic males (ZZ) Figure 1. dsRNA length knockdown dependency. (A) Effect of administered dsMr- into 'neo-females'. When crossed with regular males, such neo- IAG lengths (19-bp, 75-bp, 100-bp, and 250-bp) on Mr-IAG transcript levels in 4 dis- females produce the preferred all-male progeny.22 tinct in-vivo experiments. Negative control – non-injected group, Positive control – entire ORF injected group (518-bp). (B) A general transcript reduction pattern As in many other eukaryotic species, in several crustacean following injection of dsMr-IAG at different lengths. species the above-mentioned knockdown pathways are highly conserved and have important roles in gene transcriptional and post-transcriptional regulation, viral defense and the suppres- 0.93, respectively). A pair of dsRNA fragments of 100-bp (Serial » sion of transposable elements.29,30 The current study examined no. 100_2 and 100_5, Fig. S1A) caused 75% decrease in fi the expression of the Dicer and Argonaute gene paralogs in expression, which was signi cantly different from expression M. rosenbergii following dsMr-IAG administration with the levels in both the negative control group and the entire ORF D < aim of elucidating possible Mr-IAG knockdown pathways, group (P-value 0.034 and P-value 0.001, respectively). fi whether transcriptional, post-transcriptional or both. Attempts to improve the knockdown ef ciency of the 100-bp Gene function studies and therapeutic manipulations that fragments by covering the entire ORF sequence with a mixture use dsRNA, which trigger RNAi pathways, typically exploit of 100-bp fragments did not yield better results (See Fig. S2). A short dsRNA, like siRNA or shRNA.4,31,32 These short dsRNA similar mixture of 75-bp dsRNA fragments caused a minor molecules, however, failed to cause Mr-IAG knockdown, in our decrease in Mr-IAG transcript level, but statistically, that result preliminary work to the current study. Therefore, we sought to was similar to those of both the negative control and the entire D determine the minimum dsRNA length required to obtain ORF injected groups (P-value 0.37 and 0.178, respectively). Mr-IAG knockdown as effective as that achieved by the entire Transcription level in the control (non-injected) group was sig- fi ORF sequence (518-bp). Furthermore, we also examined the ni cantly different from that of the entire ORF injected group D possible cellular mechanism behind this manipulation and (P-value 0.012). All attempts to knockdown Mr-IAG the changes that occurred in the expression levels of Mr-IAG expression using a mixture of 19-bp, short interference RNA and several RNAi associated genes, such as the SID1 and Dicer (siRNA) did not cause down regulation compared with the neg- D fi paralogs, in response to different dsMr-IAG lengths. ative control group (P-value 1.0) and were signi cantly different from the results for the entire ORF (P-value < 0.01). In Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 Results all the distinct length dependency experiments (mixture of 19- bp, 75-bp, 100-bp or 250-bp fragments), Mr-IAG expression Effect of dsRNA fragment lengths shorter than the full ORF levels in the negative control groups and in the entire ORF fi on Mr-IAG knockdown ef ciency injected groups were significantly different. Attempts to knock down Mr-IAG expression levels using shorter dsMr-IAG fragments showed a clear correlation Presence and phylogeny of knockdown-pathway-related between dsRNA fragment length and Mr-IAG knockdown genes in M. rosenbergii efficiency, i.e., the longer the dsRNA fragment, the greater the knockdown of Mr-IAG expression levels (Fig. 1). Of the four To understand the difference in M. rosenbergii in the acti- lengths tested, only the 250-bp dsRNA fragment caused a sig- vation of RNAi and its dependence on dsRNA length com- nificant knockdown effect similar to the entire ORF (»99% pared with its activation in other RNAi pathways, we D decrease compared with the negative control, F(2,15) 18.871, examined the main RNAi associated genes, members of the P-value < 0.0001). The relative Mr-IAG expression levels while Dicer and Argonaute families. Mining our M. rosenbergii using the 250-bp fragments were significantly different from transcriptomic library resulted in multiple sequence align- that of the negative control and strikingly similar to the effects ments with the Mr-Dicer family based on putative amino of the entire ORF sequence (P-value < 0.001 and P-value D acid sequences showing cluster deviations for both Dicer1 RNA BIOLOGY 3

and Dicer2 (Fig. 2A). M. rosenbergii Dicer1 (Mr-Dicer1) homology to Ago1 orthologs from other crustacean species showed homologies to Marsupenaeus japonicus Dicer1, such as Panulirus interruptus Ago1, Mj-Ago1 and Lv-Ago1 Penaeus monodon Dicer1, Litopenaeus vannamei Dicer1 and (99%, 98% and 98%, respectively) and to Dm-Ago1 (86% Drosophila melanogaster Dicer1 (83%, 71%, 70% and 67%, homology). The cases of Argonaute2 and Argonaute3 were dif- respectively). Mr-Dicer2 showed homologies to Lv-Dicer2, ficult to differentiate by phylogenetic analysis. Homologies of Pm-Dicer2 and Dm-Dicer2 (51%, 51% and 27%, respec- 43% and 38% were noted between Mr-Argonaute paralogs of tively). Homology of 32% was found between the Mr-Dicer Mr-Ago1 to Mr-Ago2 and Mr-Ago3, respectively. A higher, paralogs. homology (48%) was found between Mr-Ago2 and Mr-Ago3. Phylogenetic analysis based on multiple amino acid To elucidate which knockdown pathway was activated as a sequence alignments of 3 Ago candidates demonstrated devia- response to dsMr-IAG manipulation, dsMr-IAG was administered tions into only two clusters: cluster A for Ago2 and Ago3, and and RNAi associated gene induction (Dicer and Ago families) was cluster B for Ago1 (Fig. 2B). Mr-Ago1 showed significant tested. Mr-IAG expression levels were significantly lower, »99% D < of control (t(10) 18.608, p-value 0.0001), as a result of dsMr- IAG injection (Fig. 3A). Comparing the expression levels of genes related to the RNAi mechanism between the group injected with dsMr-IAG to a non-injected group demonstrated that Mr-Dicer2 fi D was signi cantly upregulated in the injected group (t(6) ¡4.873, p-value < 0.01) (Fig. 2B)whileMr-Dicer1 expression levels were similar to those in the non-injected group (p-Value D 0.128). 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-Ago3 expression levels were similar to those in the non-injected group D¡ D (t-test, t(13) 1.091, P-value 0.295 and Mann-Whitney U-test, P-value D 0.779, respectively).

Mr-SID1, knockdown pathway and dsRNA length Mr-SID1, which encodes for a passive channel with specificity to dsRNA molecules, was found by comparing the L. vannamei SID1 amino acid sequence to our M. rosenbergii transcriptomic library, resulting in a transcript with 52% homology. This transcript (Mr-SID1) was fully sequenced, and 11 transmembrane regions were predicted in its putative protein sequence by a bioinformatics hydrophobicity evaluation (Fig. S3A). Phyloge- netic analysis demonstrated that SID1 is conserved between different arthropod species (Fig. S3B). With the aim of confirming the function of Mr-SID1, its expression levels were measured in response to administration of dsRNA and DNA (both of GFP sequence) and PBS as a negative control (Fig. 4A). Mr-SID1 was upregulated only in the D D presence of dsRNA (F(2,19) 7.19, p-value 0.004), showing fi

Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 the speci city of Mr-SID1 to dsRNA. Furthermore, loss of function of Mr-SID1 showed a lethal effect after two consecu- tive injections of dsMr-SID1 and dsGFP (Fig. 4B). In all the control groups, including PBS, dsGFP and dual injections of two exogenous dsRNA (dsGFP C dsRB), high survival rates were demonstrated (100%, 100% and 87%, respectively). All the dsMr-SID1 and dsGFP injected individuals died during the 24 h following the second injection. To conclude the study, expression levels of Mr-SID1 and the other components of the RNAi mechanism were tested in Figure 2. Phylogenetic analysis knockdown pathway associated genes. Phyloge- response to different fragment lengths of dsMr-IAG based on netic analysis based on multiple alignments using Mega 6.0 software. The neigh- the same length dependency experiments shown in Fig. 1. bor-joining tree was constructed according to multiple alignments of the putative amino acid sequences: (A) Dicer constructed according to phylogenetic analysis of The correlation clearly showed that dsMr-IAG fragment full amino acid sequences. (B) Argonaute phylogenetic analysis constructed length was positively correlated with Mr-SID1 upregulation according to multiple alignments of PIWI domain amino acid sequences. Tree scale (Fig. 5A). A signiﬁcant induction of Mr-SID1 expression lev- indicates the evolutionary distance by tree branch lengths, which are proportional to sequence differences. Bootstrap values represented by numbers next to the els was measured by dsMr-IAG fragment lengths of 100-bp branches. GenBank accession numbers are in Table S5. (P-value D 0.01) and 250-bp (P-value D 0.002), and this 4 N. SHPAK ET AL.

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).

activity was similar to the upregulation of Mr-SID1 expression environmental applications.3,33,34 The use of such a biotech- levels following injections of the entire ORF (P-value D nology was previously suggested to be safe based on the 0.003 and 0.027, respectively). The administration of a mix- temporal nature of the intervention and its lack of any ture of 19-bp fragments did not cause Mr-SID1 induction, overtly apparent long-term consequences.23,35 These studies whose expression levels were similar to that of the negative presented evidence based on social structure, growth perfor- control. The expression levels of Mr-SID1 following the injec- mance and masculine reproductive characters at the ana- tion of 75-bp fragments of dsMr-IAG were different from tomic and molecular levels. Moreover, our observations that those of both the negative control and the entire ORF the elapsed times until disappearance of the exogenous (P-value < 0.001, for both). dsRNA from prawn tissues and the return of Mr-IAG tran- The same correlation shown above with respect to Mr-SID1 script levels to normal during the post silencing period and different dsMr-IAG fragment lengths was also found for were comparable, suggesting that RNAi-based technology is Mr-Dicer2 with the exception of the induction caused by 100-bp temporal and indeed safe.23,35 The current study, which dsRNA fragments (Fig. 5B). The expression levels of Mr-Dicer2 examined the mechanism of the knockdown pathway, pro- in response to 100-bp fragments of dsRNA were significantly vided additional support for the temporal nature of the higher than the entire ORF injected (P-value D 0.041), and RNAi-based production of all-male cultures and rejects the those of both the 100-bp fragment and the entire ORF were sig- possible involvement of permanent or epigenetic interfer- nificantly higher than that of the non-injected negative control ence processes on the genome associated with the transcrip- group (P-value < 0.001, both). Administrations of 75-bp and tional silencing pathway. 250-bp fragments of dsRNA elicited higher Mr-Dicer2 expression Three known RNAi pathways, i.e., micro RNAs (miRNA), levels than those of the non-injected group (P-value D 0.005 short interfering RNA (siRNA) and PIWI interacting RNA and P-value < 0.001, respectively) and, in contrast to the results (piRNA), are associated with Argonaute protein family obtained for the 100-bp fragments, showed similar Mr-Dicer2 members.12 In the current study, the Dicer and Ago homo- expression levels to that obtained in response to injection of the logs were discovered and fully sequenced in M. rosenbergii. entire ORF (p-value D 0.7 and 0.98, respectively). In response The clustering of each homolog to other members from Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 to administration of the mixture of 19-bp fragments, the expres- related species further supports the correct identification of sion levels of Mr-Dicer2, similar to those of Mr-SID1, were not the distinct Mr-Dicer and Mr-Ago members allowing the affected, and its expression levels were similar to those in the examination of the response of each homolog transcript level non-injected group (p-value D 0.99). to dsMr-IAG administration. Only transcripts of Mr-Dicer2 As a reference, we used the expression levels of Mr-Dicer1 and Mr-Ago2, essential components for RNAi response and from the miRNA pathway. This gene remained at the same low post-transcriptional silencing, were significantly increased levels in all the treatments, thus demonstrating the absence of a after dsMr-IAG injection. Other transcripts in these families, correlation with dsMr-IAG fragment length (Fig. 5C). such as Mr-Ago1 and Mr-Dicer1 associated with nuclear transcriptional silencing16,17 or Ago3 associated with piRNA,21 were not affected by the introduction of dsMr- Discussion IAG. Since the Dicer2 and Ago2 proteins are known to be The Mr-IAG knockdown pathway involved in the post-transcriptional pathway where they exert temporal cytoplasmic effects,12,36 the results of the Full and functional sex reversal of male crustaceans into present study support both the temporal nature of the effects females caused by the use of RNAi demonstrates the vast of dsMr-IAG administration and the safety of its use in potential of RNAi-based biotechnologies in aquaculture and aquaculture and environmental applications. RNA BIOLOGY 5

lower the Mr-IAG transcript levels. A similar tendency was demonstrated in D. melanogaster.6 To gain insight into this phenomenon, different dsMr-IAG fragment lengths were evaluated for their effects on the expression levels of genes associated with the post-transcriptional knockdown pathway. To investigate the mechanism of action, Mr-SID1, a passive transmembrane channel that specifically delivers dsRNA into somatic cells,5,7 was sequenced and aligned to orthologs from arthropod species. Mr-SID1 is the main gate for passive dsRNA uptake into the cell and indeed its transcript levels were only upregulated in the presence of dsRNA. The results are supported by those of Shih and Hunter,7 who examined the differences in membrane conductance in SID1-expressing cells in the presence of dsRNA and DNA and showed that conductance changed only in the presence of dsRNA. Based on the amino acid homology of Mr-SID1 to other species with the same predicted 11 transmembrane regions and based on the results of the functional test of Mr-SID1, we suggest that Mr-SID1 is the dsRNA-gated channel in M. rosenbergii. Yet SID1 is not the only way known for dsRNA delivery into the cytosol. Many metazoan cells can deliver an exogenous dsRNA via receptor mediated endocytosis, an uptake mechanism that seems to be evolutionarily conserved.8 To determine whether Mr-SID1 is the main avenue for the delivery of dsRNA into M. rosenbergii cells, we attempted to knock down SID1, after which we planned to try SID1 knockdown in parallel with dsMr-IAG administration. The latter experiment, however, could not be performed since the effect of dsMr-SID1 administration was lethal unlike the cases of other documented RNAi- associated genes.40,41 It should be noted that dsMr-SID1 was designed based on a wide M. rosenbergii transcriptomic library42 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 study organism, lethality caused by off-target effects could not be completely ruled out. In addition, while we also cannot rule out the possibility that dsRNA was internalized through endocytosis, we suggest that SID1 is a major component in dsRNA traf- ficking. This is supported by results from SID1 non expressing Figure 4. Mr-SID1 expression in response to dsRNA injection and its loss of function effects. (A) dsRNA specificity demonstrated by a comparison between Mr- D. melanogaster S2 cells, in which dsRNA is endocytotically SID1expression level in dsRNA-injected compared with dsDNA-injected groups delivered,8 while the expression of SID1 in those cells increases (dsRNA and dsDNA contain the same GFP exogenous sequence) and negative con- the efficiency of gene knockdown.6 In the M. rosenbergii sys- trol, PBS-injected group (n D 7 in each group). (B) Survival rates of juvenile

Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 prawns following dsMr-SID1 injection (with dsGFP). Controls included the PBS- tem, the presence and differential activity of Mr-SID1 seem to injected group administered a double volume similar to the volume used in the suggest that it plays a crucial role in regulation of systemic dual injection of 2 dsRNAs, the dsGFP injected group, and the dsGFP and dsRB RNAi and the cell’s defense mechanism against viruses. injected group. The present study showed a positive correlation between injected dsMr-IAG fragment length and Mr-SID1 transcript Post-transcriptional knockdown pathway and dsRNA induction level. In contrast to our ﬁndings, another study that length examined C. elegans SID1 expression in D. melanogaster S2 The current study also focused on the ability to knock down cells did not ﬁnd it to be selective to dsRNA length.43 The Mr-IAG using fragments with lengths shorter than the entire authors of the latter article suggested that dsRNA length depen- ORF. Gene knockdown in various organisms was demonstrated dency probably does not occur through SID1.43 It is thus using short dsRNA fragments (siRNA) for loss of function pro- suggested that the tendency of Mr-SID1 transcripts to respond cedures, mostly in-vitro.37-39 Thus, we attempted to knock to dsRNA lengths is regulated by factors downstream, and not down Mr-IAG in-vivo using siRNA. These attempts failed, elic- upstream, of the dsRNA delivery, and that it is not self-regu- iting the need to examine the correlation between dsRNA lated by the transmembrane channel itself. It is also suggested length and knockdown success. A clear relationship between that dsRNA delivery depends on the dsRNA retention ability of dsMr-IAG lengths and Mr-IAG transcript levels was found, in the Dicer-RISC complex. Insofar as SID1 is a passive channel, which the longer the administered dsMr-IAG fragment, the dsRNA retention by Dicer-RISC is essential to allow the 6 N. SHPAK ET AL.

Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 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 experiments (Fig. 1).

intra-cellular dsRNA concentration to decrease, resulting in dsRNA molecules and that the level of induction depends on more dsRNA molecules passing into the cell through SID1 the number of molecules. Among the solutions of the different according to intra- and extra-cellular dsRNA concentrations.43 fragments at the same dsRNA concentration (in this study Mr-Dicer2 transcript levels also changed in response to 5 mg/g body weight), that with the 100-bp fragments contains dsMr-IAG length but without a clear, linear tendency as was many more molecules, and this could explain the finding that found for Mr-SID1. In the case of Mr-Dicer2, the highest upre- the highest upregulation obtained here was for Mr-Dicer2. gulation was obtained with dsRNA fragments of 100-bp compared with the other fragment sizes. Moreover, from among Concluding remarks the fragment lengths tested, only the 19-bp fragments failed to cause any Mr-Dicer2 upregulation. These results seem to con- For the first time, the Dicer and Argonaute genes were fully tradict observations of knockdown success, which was better sequenced in M. rosenbergii, which enabled us to elucidate the for fragment lengths of 250-bp and 500-bp. It is suggested that knockdown pathway of Mr-IAG, the target of the manipula- Mr-Dicer2 is influenced by the ability of SID1 to deliver the tion that causes the full and functional sex reversal of RNA BIOLOGY 7

M. rosenbergii males into females. This study showed that the The TranscriptAid T7 High Yield Transcription Kit dsMr-IAG-based manipulation manifests through a temporal, (Thermo Scientific, Lithuania) was used to generate single- post-transcriptional pathway, the well-known siRNA pathway. stranded RNA according to the manufacturer’s instructions. The temporal nature of the latter pathway further supports RNA molecules were purified by phenol-chloroform (1:1) and the safety of using such RNAi-based biotechnologies in aqua- ammonium-acetate and precipitated with ethanol. Sense and culture and environmental applications. antisense strands were hybridized by incubation at 70C for In this study, RNAi-based manipulation of the crustacean 15 min, 65C for 15 min and at RT for 30 min. dsRNA quality insulin-like androgenic hormone also demonstrated a clear was assessed on an agarose gel and diluted to 5 mg/ml. dsRNA dependency on dsRNA molecule length, such that dsMr-IAG was kept at ¡80C until used. fragment length was negatively correlated with Mr-IAG transcript level. Moreover, the induction of genes associated with the Mr-IAG knockdown attempts using fragments shorter than post-transcriptional pathway and SID1 was also differentially the entire ORF influenced according to dsMr-IAG fragment length. Unlike numerous clear cases of RNAi driven by the administration of Four distinct experiments were performed, one for each dsRNA small dsRNA fragments in-vitro, the case presented here demon- length (19-bp, 75-bp, 100-bp, and 250-bp). Each experiment strates length dependency when the experiments are performed included a non-injected group as a negative control and an in-vivo. Our results thus support the need for further study of entire dsMr-IAG ORF injected group as a positive control. It is RNAi phenomena in the context of the entire organism. important to note that the determination of which animals would constitute the non-injected control groups was based on previous studies, which also used exogenous dsRNA or DDW Materials and methods injection as described in Lezer et al.35 and in Sharabi et al.,42 – Animals with similar results. Each experimental group included 6 8 small M. rosenbergii males (Mean body weight D 12.5 g). Sin- M. rosenbergii prawns of the BGU-line were maintained at gle injections of the different dsMr-IAG lengths (5mg dsRNA/ Ben-Gurion University of the Negev (BGU) facilities as gr body weight) were performed once, and then 48 hours post- 44 described by Shpak et al. The all-male progenies originating injection, prawns were anesthetized in ice cold water, dissected ’ from crosses between males and 'neo-females were supplied by and total RNA was extracted from their androgenic glands the Tiran Shipping group through its subcontractor hatchery, using the EZ-RNA Total RNA Isolation Kit (Biological Indus- Colors Ltd., Hatzeva, Israel. This hatchery holds only genetic tries, Beit Haemek, Israel) according to the manufacturer’s males form the BGU line, and genetic females have never been instructions. cDNA was prepared in a reverse-transcriptase allowed at this farm. reaction containing 1 mg total RNA using a qScript cDNA syn- All dsRNA length dependency experiments were performed thesis kit (Quanta Biosciences, Gaithersburg, MD, USA) using small males whose morphotype was determined accord- according to the manufacturer’s instructions. Relative quantifi- 45 ing to Kuris et al. It should be noted that small males are cation of Mr-IAG expression levels was obtained as described 46 sexually active and have an active androgenic gland. in Ventura et al.22 with the FastStart Universal Probe Master fi Mr-SID1 loss of function and speci c induction of Mr-SID1 (Rox; Roche Diagnostics GmbH) and Universal ProbeLibrary by dsRNA experiments were performed using post larvae all- Probe 144 (Roche). Mr-18S (GenBank accession no. male progeny from the all-male hatchery mentioned above. GQ131934) was used as a normalizing gene and quantified using specific primers as described in Ventura et al.22 with the above-mentioned mix and Universal ProbeLibrary Probe 152 Synthesis of double-stranded RNA (Roche). Reactions were performed with the ABI Prism 7300 Eight dsRNA fragments of 19-bp and two fragments of 75-bp Sequence Detection System (Applied Biosystems).

Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 from the Mr-IAG mRNA sequence were synthesized and supplied by BioSpring, Frankfurt, Germany (Fig. S1A). Five RNAi associated gene mining dsRNA fragments of 100-bp covering approximately the entire Mr-IAG ORF and one fragment of 250-bp were synthesized in- RNAi associated-genes were mined from our composite vitro in our laboratory (Fig. S1A). A pGEMÒ-T Easy plasmid M. rosenbergii transcriptomic library, which contains more containing the Mr-IAG open reading frame sequence served as than 290 million reads assembled into 108212 contigs, fully the template for dsMr-IAG synthesis. The template was ampli- described by Ventura, et al.47 and Sharabi, et al.42 The mining fied by PCR, primed by two gene-specific primers with a T7 was done based on orthologs from related crustacean species promotor site at the 5' of one primer (T7P) (see primers and using a BLAST search to reveal Dicer and Ago homologs and T7 promotor sequences for dsRNA synthesis in Table S1). SID1sequences. M. rosenbergii-Dicer1 (Mr-Dicer1) was com- Primer pairs were as follows: the sense strand was synthesized pared with P. monodon Dicer1 (GenBank accession number using primer T7P forward vs. reverse primer, while the anti- ABR14013.1) and Mr-Dicer2 was compared with L. vannamei sense strand was synthesized by T7P reverse vs. forward Dicer2 (GenBank accession number ACF96960.1). Mr-Argo- primer. PCR amplicons were electrophoresed on a 1.3% agarose naute1 was compared with M. japonicus Argonaute1 (GenBank gel and visualized with ethidium bromide and UV light, excised accession number ADB44074.1). Mr-Argonaute2 was com- from the gel, and purified with a AccuprepÒ PCR purification pared with M. japonicus Argonaute2 (GenBank accession num- Kit (BIONEER Co., Daejeon, South Korea). ber AB665954.1). Mr-Argonaute3 was compared with P. 8 N. SHPAK ET AL.

monodon Argonaute3 (GenBank accession number the entire dsMr-IAG ORF [5mg dsRNA/gr body weight] by a AGC95229.1). M. rosenbergii-SID1 was compared L. vanna- micro injector. Androgenic glands and hepatopancreases were mei-SID1 (GenBank accession number HM234688.1). dissected for total RNA extraction, 48 h post injection as Putative sequences obtained from the library were computa- described above. Total RNA was isolated and cDNA was pre- tionally translated using the ExPASy Proteomics Server (http:// pared as described above. Mr-IAG expression levels were ca.expasy.org/tools/dna.html), and the longest open reading detected from total RNA extracted from androgenic glands to frame was selected. Transmembrane regions of Mr-SID1 were verify Mr-IAG knockdown success. Relative quantification of evaluated using the Simple Modular Architecture Research Mr-IAG expression levels was obtained as described above. Tool (SMART) (http://smart.embl-heidelberg.de/).48 Relative quantification of Mr-Dicer (Mr-Dicer1,2) and Mr-Argonaute (Mr-Ago1,2,3) expression levels were tested using the total RNA isolated from the hepatopancreas, In-vitro validation and sequencing preliminarily showing high expression levels. Mr-Dicers and Total RNA was extracted from the hepatopancreas of dsMr- Mr-Argonautes expression levels were obtained using forward IAG-injected [5mg/ml] animals, while anesthetized in ice-cold and reverse primers (Table S4) with FastStart Universal Probe water, 48 h post-injection. Total RNA was isolated and cDNA Master (Rox; Roche Diagnostics GmbH) and Universal was prepared as described above. ProbeLibrary Probes (Roche) (Table S4). For all these relative Putative Mr-Dicers (Mr-Dicer1,2),Mr-Agos(Mr-Ago1,2,3)and quantifications, Mr-18S was also quantified by real-time RT Mr-SID1 sequences were in-vitro validated by PCR using gene PCR as a normalizing gene as described above. specific primers (Table S2). The PCR products were cloned and sequenced as described by Ventura, et al.28 Mr-Dicer2 and Mr- Double-strand RNA and Mr-SID1 induction SID1 5' end were obtained by 5' rapid amplification of cDNA ends (RACE 5') performed with the Clontech SMART RACE kit To confirm the role of SID1 in M. rosenbergii, juvenile males (BD Biosciences, Palo Alto, CA) following the manufacturers (n D 22) (mean weight D 0.22 gr) were divided into three protocol. PCR was performed using a gene-specificreverse groups: phosphate-buffered saline (PBSX1) injected (n D 8), primer (Table S3) and the Universal Primers Mix (UPM) pro- DNA-GFP injected (n D 7) and dsRNA-GFP injected (n D vided in the kit. To confirm the obtained sequence, 5' RACE 7). dsRNA-GFP was synthesized as described above. DNA frag- product was amplified by PCR using a reverse nested primer ments that contained the same GFP sequence were amplified closer to the 5' end (Table S3) and a UPM nested primer, also by PCR. PCR products were purified using NucleoSpinÒ Gel provided in the kit. RACE 5' and nested 5' products were cloned and PCR Clean-up (MACHEREY-NAGEL GmbH & Co., and sequenced as described above. To ensure the quality of the Duren,€ Germany). The procedure included two injections of sequence, each region of the gene was sequenced four times. 5mg per g body weight each every 24 h. Prawns were anesthe- The 3' end of Mr-Ago3 was obtained by 3' rapid amplifica- tized in ice cold water and their hepatopancreases were dis- tion of cDNA ends (RACE) as described above using a gene- sected 24h post second injection. Total RNA was extracted and specific forward primer (Table S3) and a UPM provided in the cDNA was prepared as described above. Relative quantification kit, and the PCR products were cloned and sequenced. To con- of Mr-SID1 expression levels was performed as described above firm the obtained sequence, the 3' RACE product was amplified (for primers and probe, see Table S4). by PCR using a forward nested primer closer to the 3' end (Table S3). This product was cloned and sequenced as The effect of Mr-SID1 knockdown described above. To ensure the quality of the sequence, each region of the gene was sequenced four times. With the aim of examining the crucial role of Mr-SID1 to gene knockdown in M. rosenbergii, the effect of Mr-SID1 knockdown was studied. Forty juvenile males (mean body Phylogenetic analysis D

Downloaded by [Ben Gurion University of the Negev] at 01:27 07 November 2017 weight 0.67 gr) were divided to four groups: DDW Known sequences of Dicer and Ago from related Crustacean injected, dsGFP injected, dual injection group of dsRB and species and SID1 sequences from different species were dsGFP (two exogenous dsRNA) and dual injection group of obtained from the GenBank database. A phylogenetic neigh- dsGFP and dsMr-SID1 (the purpose of dsGFP was to induce bor-joining tree was constructed using Mega 6.0 software Mr-SID1 expression level). The procedure included two (Molecular Evolutionary Genetics Analysis, version 6.0) injections of 10mg mixed dsRNA per g body weight with according to ClustalW multiple sequence alignments of the 24 h interval and survival rates were monitored. Synthesis putative amino acid sequences of Dicer, Argonaute and SID1. of dsGFP was done as described above. dsRB,anexogenous Bootstrap values (%) of 1000 replicates were calculated for each gene, was synthesized as described by Lezer et al.35 The node of the consensus tree attained.49 synthesis of dsMr-SID1 was done as described above using speciﬁc primers (Table S1) Mr-IAG knockdown pathway determination RNAi-associated genes expression levels as a response To elucidate the Mr-IAG knockdown pathway, 15 small to different dsMr-IAG length M. rosenbergii males (mean body weight »15 gr) were divided into two groups, the non-injected group (n D 8) and the RNAi-associated gene expression levels (Mr-SID1, Mr-Dicer2 injected group (n D 7), whose members were injected with and Mr-Dicer1) were measured using the same RNA isolated RNA BIOLOGY 9

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