Identification and validation of circRNAs

We prepared total RNA samples from cultured Drosophila S2 cells that were either left untreated or treated with a combination of 20-hydroxyecdysone (20-HE) and a mixture of Escherichia coli and Micrococcus luteus, which activates innate immunity signaling [6]. To enrich circRNAs, RNA samples were subjected to ribosomal RNA depletion followed by RNase R treatment, which specifically degrades linear RNAs. Subsequently, cDNA libraries were constructed and sequenced using the paired-end strategy. CircRNAs were identified based on the presence of “back-spliced” exon junctions, in which the 3’ end of a downstream exon is linked to the 5’ end of an upstream exon. In total, ~5000 circRNA candidates were identified in various Drosophila samples (S1 Table). A significant number of circRNA candidates overlap with those identified in previous studies (S2 Table) [51,54]. Among the 1095 circRNA candidates identified from S2 cells that have been treated with a combination of 20-HE and a mixture of Escherichia coli and Micrococcus luteus, a significant proportion (37.8%, multi-exonic) contains two or more exons, whereas the majority (58.5%, mono-exonic) is composed of either the whole or partial single annotated exons (S1 Fig and S3 Table). A small fraction (2.1%, labeled as mapping to introns) appears to map exclusively to the intronic region of annotated transcripts. However, they are predominantly antisense to annotated transcripts and likely products of cryptic splicing events, since consensus splice donor and acceptor sequences (5’-GU and 3’-AG) were found to flank the region that gives rise to these circRNAs. The remaining circRNA candidates (1.6%) were collectively labeled as “miscellaneous”. So far, we have not found any intron-containing circRNAs in our validated circRNA collection. These observations suggest that intron-containing circRNAs are not abundant in Drosophila. Alternatively, it is possible that the computational strategy employed in this study, which dictates the presence of splice donor and acceptor sites in the flanking region as a prerequisite for circRNA identification, has filtered out intron-containing circRNAs.

To validate circRNAs, we conducted a combination of RT-PCR, Sanger sequencing and RNase R resistance assays. Convergent primer pairs are expected to amplify products from both genomic DNA (gDNA) and cDNA templates, whereas divergent primer pairs are expected to exclusively amplify regions that encompass the back-spliced exon junctions (S2A–S2D Fig). Second, we purified PCR products derived from the divergent primer pairs and performed Sanger sequencing, which confirmed the unique back-spliced exon junctions (S2A and S2B Fig). Third, we performed RNase R treatment, which reveals that circRNAs are resistant, whereas their linear siblings are susceptible to RNase R digestion (S2E and S2F Fig). Thus combined results from RT-PCR, Sanger sequencing and RNase R resistance analyses confirm circular configuration. Altogether, we were able to validate ~50 circRNAs (S4 Table).

The circRNA Edis regulates innate immunity in cultured Drosophila cells

To identify circRNAs relevant to innate immunity signaling, we depleted individual circRNAs in S2 cells by designing shRNAs targeting the unique back-spliced exon junctions (Fig 1A). Cells were subsequently treated with 20-HE and then either left untreated or treated with DAP-type peptidoglycan (PGN), a component of the cell wall of Gram-negative and select Gram-positive bacteria that potently activates IMD antibacterial innate immunity signaling [17]. Next, we performed RT-PCR analysis to examine the impact on levels of the Diptericin (Dpt) mRNA, which encodes a potent antibacterial peptide. This analysis identified a number of circRNAs that affected Diptericin expression (S3 Fig). For subsequent analysis, we focused on circ_1705 (circ_Ect4), which is derived from a single exon of the Ect4 transcripts (Figs 1A and S4). This circRNA was among the ones identified in previous studies [51,54]. We named it Edis (Ect4-derived immune suppressor) based on the phenotypes described below. Levels of Edis do not seem to change in S2 cells that are subjected to various combinations of 20-hydroxyecdysone treatment and/or bacterial infection (S5 Fig). Compared to controls, depletion of Edis in S2 cells by two independent shRNAs (shEdis-A and -B) leads to an increase in Dpt mRNA levels, both in the absence and in the presence of PGN treatment (Figs 1A–1C and S6A). In addition, Edis-depleted cells display enhanced Relish processing, a hallmark of IMD antibacterial signaling pathway activation (Fig 1D and 1E). Notably, levels of the linear Ect4 transcript were not affected in Edis-depleted cells (Figs 1B and S6B–S6D). Furthermore, the increase in Dpt expression elicited by Edis knockdown is alleviated by depleting components of the IMD signaling pathway such as dFADD, Dredd, DmIKK (Ird5 and Kenny) or Relish (Fig 1F and 1G). Similar results were obtained by monitoring the expression of a luciferase reporter gene driven by the promoter of the antimicrobial peptide gene Attacin-A (Att) (Fig 1H). Consistent with these findings, we detected an increase in Relish occupancy on the Dpt promoter upon Edis depletion, but not on the negative control Diedel promoter (Fig 1I), further reinforcing the notion that Edis impacts the IMD signaling pathway by regulating Relish activity. In contrast, depletion of linear Ect4 transcripts by two independent dsRNAs that do not overlap with Edis had no significant impact on Edis or Dpt expression (Figs 1A and 1J, S7). These results demonstrate that the circRNA Edis regulates innate immunity in cultured S2 cells.

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Fig 1. Edis depletion impacts innate immunity signaling.

(A) A diagram depicting the linear Ect4 mRNA and circular RNA Edis derived from the Ect4 locus as well as the mapping of various dsRNA/shRNA reagents targeting Ect4 (gray bars) or Edis (green bars). Thick and thin bars, respectively, represent exons and introns. (B-C) S2 cells stably transfected with an shRNA construct targeting the back-spliced exon junction of Edis (shEdis-A) or a control (Ctrl) shRNA against white (w) were treated with 20-hydroxyecdysone (20-HE) and subsequently with (+) or without PGN (-). Levels of linear Ect4 and circular Edis transcripts (B) or Diptericin (Dpt) mRNA (C) were measured by RT-qPCR and normalized to RpL32 (student t test, n = 3, * p<0.05; ** p<0.01; *** p<0.001; ns, non-significant). (D-E) Edis or control knockdown cells were with 20-HE and subsequently with PGN. Relish cleavage was measured by immunoblot (D) and quantified (E). Right and left panels, respectively, show fold change in Rel cleavage with and without PGN treatment (student t test, n = 3, * p<0.05). (F-G) S2 cells stably transfected with two independent shRNAs targeting Edis (shEdis-A or -B) or control sh-w construct were subsequently transfected with various dsRNAs (below). Cells were treated with 20-HE and levels of Dpt mRNA (F) and Edis (G) were measured (student t test, n≥3, * p<0.05; ** p<0.01; *** p<0.001). (H) S2 cells stably transfected with the shEdis or control shRNA construct, were transfected with the Att-luc reporter driven by the antimicrobial peptide gene Attacin promoter and the pActin-Renilla control reporter constructs together with various dsRNAs against various components of IMD signaling. Cells were treated with 20-HE and reporter gene activities were measured and normalized (student t test, n≥5, *** p<0.001). (I) S2 cells stably transfected with the shEdis or control shRNA construct were transfected with the Flag-Relish and control HA-Ran expression constructs. Cells were treated with copper to induce transgene expression, and subjected to ChIP assay using anti-Flag antibody. Enrichment of DNA fragments from the DptA and Diedel (control) promoters was measured (one-way ANOVA, n = 2, * p<0.05; ** p<0.01; ns, non-significant). (J) S2 cells were transfected with dsRNA targeting exclusively the linear Ect4 transcript or a control dsRNA targeting LacZ. Cells were treated with 20-HE and levels of Ect4, Edis and Dpt were measured (student t test, n = 3, *** p<0.001; ns, non-significant).

https://doi.org/10.1371/journal.pgen.1010429.g001

Next, we examined the impact of Edis overexpressing on the IMD pathway in S2 cells [62]. Overexpression of Edis, but not a control circRNA circ_714, causes a strong reduction in the activity of the Att-luc reporter and/or Dpt mRNA levels both in the presence and in the absence of PGN treatment (Figs 2A and 2B, S8). In addition, Relish cleavage, elicited by either PGN treatment (Fig 2C and 2D) or ectopic expression of Dredd (Fig 2E and 2F), the caspase that carries out Relish endoproteolytic processing, is significantly compromised by Edis overexpression. Furthermore, we found that Edis overexpression mitigates the activation of both Dpt expression (Fig 2G and 2H) and the Att-luc reporter gene (Fig 2I) by ectopic expression of select components of IMD signaling (full length PGRP-LC or IMD, or constitutively active forms of dTAK1 (TAK1Δ) or Relish (RelΔS29-S45)) [22,23]. Lastly, upon restoration of Edis expression in Edis-knockdown cells, the AMP hyperactivation phenotype is suppressed (Fig 2J and 2K). Note that the exogenous Edis product from the circRNA expression construct contains back-spliced exon junction sequences derived from the vector that are distinct from those in the endogenous Edis. Therefore, the exogenous Edis is shRNA-resistant. Importantly, the linear Ect4 transcript levels remain unchanged (Fig 2A and 2K). We conclude that the circRNA Edis suppresses innate immunity signaling in cultured S2 cells.

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Fig 2. Edis overexpression blocks IMD innate immunity signaling.

(A-D) S2 cells were transfected with an Edis expression construct or empty vector, together with the Att-luc reporter and the pActin-Renilla control reporter. Cells were treated with 20-HE and PGN. Levels of Ect4 and Edis (A, student t test, n = 3, *** p<0.001; ns, non-significant), normalized reporter activity (B, student t test, n = 3, ** p<0.01; *** p<0.001), and Relish processing (C) are shown. Quantification of Relish processing immunoblot in C is shown in D (student t test, n = 3, * p<0.05; *** p<0.001). (E-F) A stable S2 cell line expressing Myc-tagged Dredd under the control of the copper-inducible metallothionein promoter was transfected with either the expression vector for Edis or the control empty vector. Cells were first treated with and 20-HE. Subsequently, cells were treated with copper to induce transgene expression, or left untreated. Relish processing was measured by immunoblot (a representative image of three independent experiments is shown). Quantification of Relish immunoblot is shown in F (student t test, n = 3, * p<0.05). (G-H) S2 cells were transfected with various combinations of expression constructs for IMD signaling components (below), together with Edis overexpression or empty vectors. Cells were treated with 20-HE and levels of Dpt mRNA (G) and Edis (H) were measured (student t test, n = 4, * p<0.05; ** p<0.01; *** p<0.001). (I) S2 cells were transfected with various combinations of expression constructs for IMD signaling components (below), together with Edis overexpression or empty vectors and the Att-luc reporter and the pActin-Renilla control reporter constructs. Cells were treated with CuSO₄ and 20-HE and reporter activity was measured and shown (student t test, n≥4, * p<0.05; ** p<0.01; *** p<0.001). (J-K) Various combinations of Edis overexpression, shRNA and empty vectors, together with the Att-luc reporter and the pActin-Renilla control reporter constructs were transfected into S2 cells. Cells were first treated with CuSO₄, and subsequently with 20-HE and PGN. Reporter activity (J, student t test, n = 6, * p<0.05; ** p<0.01; *** p<0.001) and levels of Ect4 and Edis (K, student t test, n = 3, *** p<0.001; **** p<0.0001) were measured. Note that the ectopically expressed Edis harbors a back-spliced exon junction that is distinct from that in its endogenous counterpart, therefore, is resistant to shRNA-mediated knockdown.

https://doi.org/10.1371/journal.pgen.1010429.g002

Edis regulates innate immunity in vivo

To further assess the role of Edis in vivo, we generated shRNA transgenic lines targeting Edis [63] and silenced Edis expression by crossing shEdis animals to ubiquitously expressed driver lines, including the medium strength daughterless-Gal4 (da-Gal4), and the strong Actin5C-Gal4 and Tubulin-Gal4 drivers. Global depletion of Edis by Actin5C-Gal4 or Tubulin-Gal4 causes lethality, whereas Edis knockdown by da-Gal4 yields a low number of progeny flies. To bypass the developmental lethality associated with Edis knockdown, we employed the da-Gal4; tub-Gal80^ts composite line [43]. At the permissive temperature (18°C), Gal80 inhibits Gal4 and shuts off the shRNA transgene; whereas at the restrictive temperature (29°C), Gal80 is inactivated, thereby allowing for the expression of the shRNA transgene. Fly crosses were kept at 18°C. Upon eclosion, age-matched progeny of appropriate genotypes was shifted to and maintained at 29°C for 5–7 days to allow for shRNA transgene expression and target gene silencing. This strategy allowed us to achieve reproducible knockdown of Edis in adults, while levels of the linear Ect4 transcript remain unaltered (Fig 3A). A control cross was set up using a shRNA transgenic line targeting gfp. Consistent with a recent report, we detected higher levels of Edis in flies kept at 29°C than those at 18°C (S9 Fig) [51]. Compared with controls (tub-Gal80^ts; da>shGFP), depletion of Edis in vivo leads to markedly elevated levels of the Dpt transcript under both non-infection and E. coli infection conditions (Fig 3B). This is consistent with the phenotype observed in S2 cells (Figs 1B and 1C, S6A). Next, we employed a similar strategy to deplete the linear Ect4 transcript using an shRNA transgene and a long hairpin dsRNA transgene that exclusively target the linear Ect4, but Edis (Fig 1A). We found that global Dpt gene expression upon Ect4 knockdown remains comparable to that in controls (Figs 3C and S10). These results demonstrate that the circRNA Edis impacts innate immunity in vivo.

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Fig 3. Edis regulates innate immunity in vivo.

(A-B) Flies carrying the ubiquitously expressed daughterless-Gal4 driver and a temperature-sensitive Gal80 were crossed to UAS-shEdis or control shGFP flies. Fly crosses were kept at 18°C. Adult progeny were shifted to 29°C for 5 days to allow for shRNA transgene expression. Flies were either left untreated or infected by E. coli, total RNAs were extracted 6-h post-infection and levels of linear Ect4 and circular Edis RNA (A) and Dpt (B) were measured (student t test, n≥3, * p<0.05; *** p<0.001; ns, non-significant). (C) The UAS-shEct4 was employed to knockdown the linear Ect4 RNA in vivo using a similar strategy as in A. Levels of Ect4, Edis and Dpt were measured (student t test, n = 3, ** p<0.01; ns, non-significant). (D) Edis knockdown and control flies in multiple groups (20–25 flies per group) were injected with a concentrated culture of Erwinia carotovora carotovora 15 (Ecc15) or PBS (control). Fly survival was recorded daily and plotted (log rank test, n≥3, * p<0.05). (E) At various time points post Ecc15 infection, groups of 4 flies were homogenized in sterile PBS. Fly homogenates were diluted and plated onto Ampicillin-LB plates, and the resultant colonies were counted. Shown are colony-forming units (CFUs) per fly (student t test, n≥4, * p<0.05). (F-G) The UAS-Edis transgenic line was employed to overexpress Edis in vivo using a similar strategy as in A. The UAS-laccase 2 empty vector transgene was included in the crossing scheme as a control. Flies were either left untreated or infected by E. coli, total RNAs were extracted 6-h post-infection, and levels of Edis (F, student t test, n = 3, * p<0.05) and Dpt (G, student t test, n = 6, * p<0.05; ** p<0.01) transcripts were measured. (H) Male da>shEdis; Gal80^ts flies and control da>shGFP; Gal80^ts flies were kept at 29°C for indicated period of time (below). Flies in groups of 15 were placed into conical culture tubes and tapped to the bottom, and the percentage of flies that can climb over the 2-centimeter mark within 15 seconds was recorded and shown (student t test, n≥4, * p<0.05; ** p<0.01; *** p<0.001). (I) Male da>shEdis; Gal80^ts flies and control da>shGFP; Gal80^ts flies in multiple groups of 25 were kept at 29°C. Fly survival was recorded daily and plotted (log rank test, n≥6, **** p<0.0001). (J) Total RNA was prepared from either fly head or torso samples, and levels of linear Ect4 and circular Edis RNA were measured (student t test, n = 3, *** p<0.001). (K) Levels of linear Ect4 and circular Edis transcripts in RNA samples from heads of young (3d) and old (23d) flies were measured (student t test, n = 3, ** p<0.01).

https://doi.org/10.1371/journal.pgen.1010429.g003

To further define the functional relevance of Edis in innate immunity, we injected either sterile PBS or concentrated culture of the fly pathogen Erwinia carotovora carotovora strain 15 (Ecc15) into Edis-depleted or control animals and monitored fly survival. While the survival of all flies displayed a decrease following Ecc15 infection, Edis-depleted animals present a significantly higher survival rate than controls (Fig 3D) that correlates with AMP expression levels. To further examine whether enhanced survival is attributable to improved pathogen clearance or immune tolerance, we monitored pathogen load at various time points post-infection. This analysis revealed that Edis-depleted animals out-performed controls in clearing Ecc15 (Fig 3E). We conclude that Edis-depletion leads to enhanced antibacterial defense in vivo.

Next, we examined the impact of ectopic Edis expression on innate immunity in adult flies using by crossing da-Gal4; tub-Gal80^ts flies to UAS-Edis or UAS-empty vector (control) animals. We found that Edis overexpression leads to a reduction in levels of the Dpt transcript under both non-infection and E. coli infection conditions (Fig 3F and 3G). Taken together, these observations solidify the notion that Edis plays an important role in suppressing innate immunity in vivo.

Neuronal Edis impacts innate immunity and animal survival

We noticed that da>shEdis animals show progressively impaired locomotor activity over time as compared to age-matched controls (Figs 3H and S11A) and a shorter lifespan (Figs 3I and S11B). To further investigate the physiological basis of this phenotype, we examined Edis expression in different tissues (S12A Fig). We also detected 9-fold higher levels of Edis in the fly head than in the torso (Fig 3J). In addition, our analysis reveals that in fly heads, levels of Edis, but not its linear sibling Ect4, display a dramatic increase with age (Fig 3K). As circRNAs are generally more stable than their linear siblings, the apparent increase in levels of circRNA over age could be due, at least in part, to differences in stability of circular and linear RNAs. These findings are consistent with recent reports showing that select circRNAs are enriched in the nervous system and further regulated by age [51,54].

To further explore the functional importance of neuron-derived Edis, we first overexpressed Edis in neurons using the pan-neuronal Elav-Gal4 driver (c155) and found it potently blocked Dpt expression (Fig 4A). Next, we depleted Edis in neurons by crossing Elav-Gal4 and shEdis flies. Remarkably, neuronal Edis knockdown by two independent shRNAs is sufficient to cause elevated levels of a panel of AMP transcripts in RNA samples prepared from fly heads (Figs 4B and S13A). Importantly, the immunity phenotype in Elav>shEdis flies was rescued by restoring Edis expression in neurons (Fig 4C), and increased Dpt gene expression is blocked in Relish null mutants (Rel^E20/Rel^E38) compared with Relish heterozygous animals (Rel^E20/+) (Fig 4D). This is consistent with the observation in S2 cells that elevated AMP gene expression upon Edis knockdown depends on Relish (Fig 1F–1H). Moreover, flies with neuronal Edis depletion similarly display significantly impaired locomotor activity that deteriorates rapidly with age. Notably, male Elav-Gal4>shEdis flies already show severe locomotor activity defects at a young age (2-5d) (Fig 4E), much earlier than that observed in female counterparts (~14d, S11C Fig), possibly due to dosage-compensation of X-linked Elav-Gal4 and/or differences in both Edis levels and knockdown efficiency in males and females (S14 Fig). Furthermore, Elav>shEdis flies display a shortened lifespan compared to controls (Figs 4F and S11D). To confirm these findings, we crossed shEdis flies to a second neuron-specific driver, nSyb-Gal4. We found that the nSyb>shEdis animals phenocopy Elav>shEdis flies (Fig 4G–4I). In contrast, glia-specific depletion of Edis does not impact innate immunity gene expression (S12B Fig). It is currently unclear whether glia-specific depletion of Edis impacts animal behavior or survival.

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Fig 4. Neuron-specific Edis depletion leads to hyperactivation of innate immunity, impaired locomotion and shortened lifespan.

(A) The UAS-Edis or control UAS-empty vector transgenic flies were crossed to the Elav-Gal4 driver line. Total RNA was prepared from dissected fly heads, and levels of the circular Edis RNA or Dpt mRNA were measured (student t test, n = 3, * p<0.05; *** p<0.001). (B) The UAS-shEdis or control UAS-shGFP flies were crossed to the neuron-specific Elav-Gal4 driver line. Levels of the Ect4, Edis or Dpt in fly head samples were measured (student t test, n = 3, * p<0.05; *** p<0.001). (C) The UAS-Edis or control UAS-laccase 2 empty vector transgenes were introduced into the Elav>shEdis flies. Total RNA was prepared from dissected fly heads, and levels of the circular Edis RNA or Dpt mRNA were measured (student t test, n = 3, *** p<0.001). (D) The UAS-shEdis flies were crossed to the Elav-Gal4 driver line. Female progeny either in the Relish heterozygous (Rel^E20/+) or trans-heterozygous (Rel^E20/Rel^E38) background were collected at ~3 weeks of age, and levels of Dpt in fly heads were measured (student t test, n = 3, *** p<0.001). (E) Locomotor activity of 2-5d old Male Elav>shEdis and control flies were measured and shown (student t test, n≥4, *** p<0.001). (F) Survival of male Elav>shEdis and control flies were measured and shown (log rank test, n = 4 to 8, **** p<0.0001). (G-I) The UAS-shEdis or control UAS-shGFP flies were crossed to a second neuron-specific nSyb-Gal4 driver line. Level of Dpt RNA in fly heads (G, student t test, n = 3, * p<0.05), climbing activity (H, student t test, n≥3, *** p<0.001) and lifespan (I, log rank test, n = 4 to 11, **** p<0.0001) of female progeny are shown. (J) The UAS-dsEct4, UAS-shEdis or control UAS-shGFP flies were crossed to the neuron-specific Elav-Gal4 UAS-Dcr-2 driver line. Levels of Ect4, Edis and Dpt transcripts were measured (student t test, n = 3, n = 2 for Elav>shEdis samples for measuring Dpt levels, * p<0.05; ns, non-significant).

https://doi.org/10.1371/journal.pgen.1010429.g004

Consistent with the report that Ect4 mutants display an increase in AMP gene expression in the tracheal airway epithelium [64], trachea-specific depletion of Ect4 led to elevated Drosomycin-GFP reporter activity (S15 Fig). To examine the potential role of Ect4 in neurons, we depleted Ect4 using the Elav-Gal4 driver. We actually detected a decrease in Dpt mRNA levels in Ect4-depleted fly heads, whereas the opposite phenotype was observed upon Edis depletion (Figs 4J and S16). The opposing roles of Ect4 in innate immunity in different cell types (neurons and trachea) may explain the lack of significant changes in global AMP gene expression in RNA samples prepared from whole flies with ubiquitous Ect4 depletion (Figs 3C and S10). Taken together, these results show an essential and potent role of neuron-derived Edis in regulating innate immunity and animal survival.

Edis is essential for neural development

Given the strong mobility and viability phenotypes associated with neuronal Edis depletion, we next investigated its effect on brain anatomy. Brains from young (4-6d) Elav>shEdis males or controls were dissected and stained with anti-Fasciclin II (FasII) antibodies, which highlight the mushroom bodies (MBs), a distinct brain structure involved in learning and memory [65–70]. Notably, in young fly brains depleted of Edis, MBs display a spectrum of severe morphological defects (missing, split and merged MB lobes) (Fig 5A and 5B). In addition, depletion of Edis in the eye using GMR-Gal4 leads to defective ommatidium structure, which further deteriorates with age (Fig 5C and 5D). Furthermore, as giant fiber is another neuronal structure with stereotypical morphology that is easy to visualize, we examined the impact of Edis depletion on grant fiber morphology. We found that Edis depletion in a subset of neurons using ok307-Gal4 results in abnormal giant fiber morphology and a decrease in dendritic area (Fig 5E and 5F). Moreover, Elav-shEdis larvae show substantial wiring defects of the neuromuscular junctions (NMJs)—a significantly higher frequency of ectopic synaptic formation from muscle 12 to muscle 13 comparing with control larvae (Fig 5G and 5H). We did not detect any significant changes in bouton numbers in the muscles.

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Fig 5. Neuron-specific depletion of Edis causes abnormal axonogenesis and neurodevelopment.

(A) Confocal images of dorsal anterior regions of adult brains with neuron-specific expression of control shgfp (left panel) or shEdis (middle and right panels) driven by Elav-Gal4 driver. In control brain (left panel), anti-FasII antibody delineates the central complex (CC) as well as the vertical α and horizontal β and γ lobes of the mushroom bodies (MBs), with γ lobes showing weaker FasII signal, as indicated. Depletion of Edis resulted in a spectrum of severe, age-dependent morphological defects in the MBs. In middle panel, one α lobe and one β lobe were mostly missing, and the remaining α and β lobes showed abnormal morphology. In right panel, both α lobes were largely missing, while the two β lobes crossed the midline and merged together. Scale bar: 20 μm. (B) Quantification of MB morphology phenotypes shown in A (Chi-squared test, sample numbers are shown on top, *** p<0.001). (C) To deplete Edis in the eye, GMR-Gal4 was used to drive shEdis transgene expression. The adult eyes of Ctrl and GMR>shEdis flies were analyzed by phalloidin staining (red) at day 5 or day 30 after eclosion. Scale bar: 10 μm. (D) Images of the 5-day- or 30-day-old fly eyes with the indicated genotypes were quantified and shown as the percentage of ommatidia with the indicated numbers of rhabdomeres per ommatidia (n = 3–5). (E) To probe the role of Edis in the giant fiber system, ok307-Gal4 was used to drive shEdis transgene expression, the giant fiber system was revealed using a membrane-bound mCD8-eGFP reporter (green). The dendritic area of giant fiber system was highlighted with dotted boxes. Scale bar: 20 μm. (F) Quantification of the average total dendritic area covered by giant fiber system dendritic termini (student t test, n = 9–13; ****, p<0.0001). (G) Representative confocal images of muscle 12 (M12) and muscle 13 (M13) NMJs of control (Elav>shGFP) and Edis knockdown (Elav>shEdis) 3rd instar larvae. The top panels show nerve terminal (anti-HRP) and synaptic vesicles (anti-DVGlut), while the bottom panels show over-saturated HRP signals for the same image to highlight muscle 6, 12 and 13. Arrow indicates an ectopic synapse formed at M13 as an extension from the M12 NMJ in Elav>shEdis animals. Scale bar = 20 μm. (H) Quantification of percentage of hemisegments in control and Edis knockdown larvae that show ectopic synapses at muscle 13 (student t test, ~10 hemisegments from each animal were scored. Nine larvae from each genotype were analyzed, **** p<0.0001).

https://doi.org/10.1371/journal.pgen.1010429.g005

Since Ect4 and Edis arise from the same gene locus, they carry identical nucleotide sequences except for the back-spliced exon junction that is unique to Edis. Thus it is of high importance to ensure the specificity of the RNAi reagents employed in our study. To this end, we have employed independent shRNA/dsRNA reagents that specifically target Ect4 or Edis (Fig 1A). It is unlikely that the innate immunity and neurodevelopment phenotypes in Edis knockdown animals are due to off-target effects, because 1) Two independent shRNA transgenes against Edis generate similar phenotypes both in vivo and in cultured S2 cells (Figs 1B, 1C, 4B, 5A and 5B, S6A and S13); 2) restoring Edis expression in neurons rescues both the innate immunity hyperactivation and MB morphology phenotypes of Elav-shEdis flies (Figs 4C, 7E and 7F); 3) our shRNA constructs against Edis do not cause significant off-target effects (S5 Table and S6B–S6D Fig), and do not impact Ect4 mRNA levels (Figs 1B, 2K, 3A and 4J, S6B, S6C, S16A and S16B); and 4) while due to lack of anti-Ect4 antibodies we could not assess the potential impact of Edis depletion on translation of the endogenous Ect4 mRNA, we found that Edis depletion did not affect the production or stability of Flag-Ect4 (S17 Fig).

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Fig 7. Edis-p rescues the innate immunity and neurodevelopment phenotypes elicited by Edis depletion.

(A-C) Stably transfected S2 cells carrying the pMT-Gal4 and UAS-shEdis or UAS-sh-w (control) constructs were transfected with empty vector or an expression construct encoding shEdis-resistant Flag-tagged Edis-p. Cells were either left untreated or treated with PGN and levels of the Dpt (A) and Edis (B) transcripts were measured by RT-qPCR (student t test, n = 3, ** p<0.01; *** p<0.001; ns, non-significant). (C) An anti-Flag immunoblot shows Fag-Edis-p expression in samples from A. Non-specific bands are labeled with asterisks. (D) Confocal images of dorsal anterior regions of adult brains with various MB morphology phenotypes. Depletion of Edis in neurons resulted in a spectrum of severe, age-dependent morphological defects in the MBs as noted above each panel. (E) MB morphology phenotypes were quantified in 3-5d old flies with neuron-specific expression of control shgfp or shEdis, together with an UAS-Edis, UAS-Flag-Edis-p or empty vector transgene, driven by the Elav-Gal4 driver (Chi-squared test). Sample numbers and percentage of normal MB in each genotype are shown (* p<0.05; *** p<0.001). (F) Climbing activity of 3d- and 30d-old flies of the same genotype as in E is shown (student t test, n = 2–5, * p<0.05; ** p<0.01; *** p<0.001; ns, non-significant). (G) A model of the proposed mechanism of function of Edis in the IMD antibacterial innate immunity signaling pathway.

https://doi.org/10.1371/journal.pgen.1010429.g007

Ect4 mutants display severe defects in neuron development, morphology and cell death upon injury [71]. In our hands, flies with neuronal Ect4 depletion (Elav>shEct4 and Elav>dsEct4) do not display defects in either mobility or MB morphology. We note the moderate Ect4 knockdown efficiency in these flies (Figs 4J and S16C–S16E). It is possible that the surviving Ect4 knockdown animals are “escapers” with a moderate degree of Ect4 depletion, as more efficient knockdown may cause lethality. Nonetheless, we detected a consistent decrease in Dpt RNA levels in both Elav>shEct4 and Elav>dsEct4 animals, which is opposite to the phenotype of Elav>shEdis animals (Figs 4J and S16C–S16E). These results suggest that Ect4 and Edis seem to positively and negatively, respectively, regulate innate immunity gene expression in neurons. On the other hand, we note that trachea-specific depletion of Ect4 led to elevated expression of AMP genes (S15 Fig), suggesting that Ect4 represses innate immunity gene expression in the trachea. These observations are consistent with the phenotypes previously reported for Ect4 mutants [64]. Thus our experimental settings allowed us to achieve a functionally relevant extent of Ect4 knockdown in the trachea. It is unclear whether Edis impacts AMP gene expression in the trachea. We conclude that the immunity and neurodevelopmental phenotypes of Elav>shEdis animals are not due to off target effects (by affecting Ect4 expression), and that Ect4 appears to differentially regulate AMP gene expression in neurons and trachea. It has been suggested that the Tollo/DNT1/Ect4 axis negatively impacts AMP gene expression in trachea [64]. The mechanism underlying the role of Ect4 in regulating AMP gene expression in neurons remains to be elucidated.

Edis encodes a functional protein

Select circRNAs can be translated into functional proteins [59,60]. We therefore explored the protein-coding potential of Edis. Edis harbors several potential opening reading frames (ORFs). The longest one spans across and terminates immediately downstream of the back-spliced exon junction. It potentially encodes a protein of 254 amino acids (Edis-p), which overlaps with the C-terminus of the Ect4 protein (Fig 6A). Next, we expressed in S2 cells an Edis minigene that contains a 3XFlag epitope tag immediately upstream of the stop codon (Fig 6B). Mass spectrometry analysis of immune-purified anti-Flag complex identified five independent peptides derived from Edis-p, thereby demonstrating the protein-coding potential of Edis (Fig 6C). We conclude that Edis can encode a protein, at least in the transgene setting.

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Fig 6. Edis encodes a functional protein.

(A) Schematic of the Ect4 and Edis-p proteins. Positions of the SAM (sterile alpha motif) and TIR (Toll/interleukin-1 receptor homology) domains are shown. (B) Schematic of the Edis minigene driven by the metallothionein promoter. The longest ORF, which encodes a potential protein product (Edis-p) derived from Edis, and the corresponding UTR are shown in green and blue, respectively. Positions of the initiation codon and stop codon are noted. A 3X FLAG epitope tag-coding sequence was placed immediately upstream of the putative stop codon. (C) Lysate from stably transfected S2 cells carrying the constructs described in B was subjected to anti-FLAG IP followed by mass spectrometry. Five independent peptides (in green) derived from the Edis-p were identified. (D) S2 cells were transfected with empty vector or the expression construct encoding Flag-tagged Edis-p. Cells were first treated with copper and 20-HE. Subsequently, cells were treated with PGN or left untreated. Levels of the Dpt transcript were measured by RT-qPCR (student t test, n = 3, * p<0.05; *** p<0.001). (E-F) S2 cells were transfected with empty vector or the expression construct encoding Flag-tagged Edis-p. Cells were first treated with copper and 20-HE. Subsequently, cells were treated with PGN or left untreated. Cell lysates were subject to immunoblot using anti-Relish (upper panel) or anti-Flag (lower panel) antibodies. Quantification of Relish immunoblot in E is shown in F (student t test, n = 3, * p<0.05; ** p<0.01). (G) Lysates from stably transfected S2 cells expressing either Flag-Edis-p or Flag-RpL22 (control) were subjected to anti-Flag immunoprecipitation. Subsequently, the immune-purified anti-Flag samples, together with input lysates, were analyzed by immunoblot in parallel using antibodies against Relish (upper panel) and Flag (lower panel). Non-specific bands (asterisks), heavy (H) and light (L) chains are also labeled. (H) S2 cell lysate (containing endogenous Dredd and Relish) was incubated with various combinations of purified recombinant Flag-Edis, Flag-Ran and Flag-Dredd in a Relish processing reaction at 30°C for 2h. The reactions were subsequently subjected to immunoblot analysis using anti-Relish and anti-Flag antibodies. (I) Quantification of Relish processing in H (student t test, n = 3, * p<0.05; *** p<0.001).

https://doi.org/10.1371/journal.pgen.1010429.g006

Next, we expressed Flag-tagged Edis-p in S2 cells and examined the impact on innate immunity. We found that ectopic expression of Edis-p phenocopies Edis overexpression, as it blocks PGN-induced Dpt gene activation (Fig 6D) and Relish cleavage (Fig 6E and 6F). In addition, we detected endogenous Relish in immune-purified Flag-Edis-p, but not the control Flag-RpL22 complexes (Fig 6G), suggesting that Edis-p interacts (either directly or indirectly) with Relish. This is further supported by the co-localization between epitope-tagged Relish and Edis-p in S2 cells (S18 Fig). However, epitope-tagged Relish does not co-immunoprecipitate with Edis-p. It is possible that the Relish-Edis-p interaction is unstable. Alternatively, the epitope tag on Relish could hinder such interaction. Nonetheless, given these observations, the simplest explanation is that Edis-p blocks Relish processing by sequestering Relish. This is consistent with our findings that the AMP gene hyperactivation phenotype elicited by Edis knockdown is suppressed by depletion of dFADD, IKK, and Dredd (Fig 1F–1H), and that Edis blocks innate immunity activation caused by ectopic expression of PGRP-LC, IMD or TAK1Δ (Fig 2G–2I), as all these IMD pathway components play key roles in Relish activation. In line with this notion, we found that purified recombinant Flag-Edis-p, but not the control protein Flag-Ran, compromises in vitro Relish processing catalyzed by both endogenous and exogenous Dredd (Fig 6H and 6I) [72]. Importantly, Edis-p suppresses the AMP gene hyperactivation phenotype elicited by Edis depletion in cultured S2 cells (Fig 7A–7C).

Next, we examined whether Edis-p can rescue the neurodevelopmental defects in Edis-depleted animals. We found that restoring Edis expression in Elav>shEdis neurons significantly rescued the MB neurodevelopmental defects. Specifically, the percentage of brains showing normal MB morphology dramatically increased to 81% upon restoring Edis levels, whereas only 42% of Elav>shEdis brains display normal MB morphology (Fig 7D and 7E). Importantly, the proportion of brains showing normal MB morphology was enhanced to 72% upon Edis-p expression (Fig 7E). Similarly, defects in mobility (Fig 7F) were also rescued by expressing Edis or Edis-p. We note that while Edis-p can rescue the MB neurodevelopmental defects in Edis-depleted neurons, the extent of rescue is milder than that of the circular RNA form of Edis (Fig 7E). Nonetheless, these observations suggest that during neurodevelopment, Edis functions at least in part, via the encoded protein Edis-p. However, we cannot exclude the possibility that Edis may also operate through other mechanisms. We conclude that Edis encodes a functional protein that suppresses innate immunity and regulates neurodevelopment by compromising Relish processing.

Statistical analyses

All statistical analyses in this manuscript were performed using biological replicates and the sample number (n) is shown for each dataset in the corresponding legend. Most analyses were performed using student t-test, except for survival experiments, which involved the log-rank test, the mushroom body morphology experiments, which involved Chi-squared test, and ChIP analysis, which involved ANOVA test. We note that our sample numbers are not high. In particular, in RT-qPCR assays sample numbers are generally equal to or slightly higher than 3. In our datasets, variations (standard deviation/standard error of the mean) are generally low among biological replicates. Unless noted otherwise, data is shown in this manuscript as mean + standard errors of the mean (SEM), * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

DNA constructs and antibodies

shRNA construct against Edis: DNA fragments containing the shEdis sequence were amplified by PCR from a DNA oligo and cloned into the Valium 20 vector using NheI and EcoRI restriction sites, to generate Valium 20-shEdis. Overexpression construct for Edis in S2 cells: a DNA fragment containing the Ect4 exon sequence that gives rise to Edis was amplified by PCR and cloned into the Hy_pMT Laccase2 MCS exon vector [62], using NheI and SacI restriction sites, to generate Hy_pMT Laccase2-Edis. Overexpression construct for Edis in flies: the Xho/T4 DNA polymerase/NotI fragment from Hy_pMT Laccase2-Edis was ligated to pUAST vector that are sequentially treated with EcoRI/T4 DNA polymerase/NotI, to generate pUAST-Edis. A similar strategy was employed to generate the control vector, pUAST-Laccase2 MCS exon vector. Expression vector for Flag-Edis-p: a DNA fragment encoding Flag-tagged Edis-p was amplified by PCR and cloned into pRmHa-3 using EcoRI and BamHI restriction sites, to generate pRmHa-3-Flag-Edis-p. We also generated an shRNA-resistant pRmHa-3-Flag-Edis-p construct in which the 3’ end of the Edis-p ORF was modified at the nucleotide level to encode the same amino acid sequence, for the rescue experiment. A DNA fragment containing the full length Relish ORF was amplified by PCR and cloned into pRmHa-3-Flag using EcoRI and SalI sites to generate the Flag-Relish expression construct. DNA fragments containing Flag- and HA-tagged Ran ORF, respectively, were amplified by PCR and cloned into pRmHa-3 using EcoRI and BamHI sites to generate Flag- and HA-Ran expression constructs. A DNA fragment containing Flag-RpL22 ORF was amplified by PCR and cloned into pRmHa-3 using EcoRI and KpnI sites to generate the Flag-RpL22 expression construct. All constructs were verified by sequencing. Primary antibodies employed in this study include anti-Flag (mouse anti-Flag, F3165, Sigma; rabbit anti-Flag, F7425, Sigma; mouse M2 anti-Flag conjugated agarose beads, A2220, Sigma), rabbit anti-HA (C29F4, Cell Signaling) and anti-Relish antibodies (21F3, Developmental Studies Hybridoma Bank, developed by Dr. Dan Hultmark). Secondary antibodies employed in this study include goat anti-mouse IgG antibody HRP conjugate (12–349, Millipore) and goat anti-rabbit IgG antibody HRP conjugate (12–348, Millipore).

Dsrna synthesis

Templates for generating dsRNAs were either requested from the DRSC (flyRNAi.org) or amplified by PCR in house. Double-stranded RNAs were synthesized using Megascript in vitro transcription kit (Ambion) and purified using RNeasy column (Qiagen).

Chromatin immunoprecipitation

Drosophila S2 cells stably transfected with sh-w, shEdis-A or shEdis-B were transfected with pMT-Flag-Relish plasmid. Cells were treated with copper sulfate for 5 days 24 hours post transfection. The cells were washed with PBS once, then crosslinked with 1% methanol-free formaldehyde for 10 minutes at room temperature. Glycine was added into the cells at a final concentration of 125 mM. Cells were incubated at room temperature for 5 minutes. Cells were subsequently rinsed with 10 mL cold PBS for 3 times, and lysed in 750 μL ChIP lysis buffer (50 mM HEPES, pH7.5, 140 mM NaCl, 1 mM EDTA, pH8.0, 1%Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, protease inhibitors) for 10 minutes. Cells were then sonicated for 15 times for 30 seconds at “High” setting (Bioruptor 300, Diagenode). After sonication cell lysate was centrifugated for 10 minutes at 4°C at a speed of 8000 g. Fifty μL of supernatant aliquot was saved at -20°C as input samples. The remaining supernatants were diluted at 1:10 with RIPA buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 2 mM EDTA, pH8.0, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, protease inhibitors). Next 40 μL anti-flag M2 affinity gel (Sigma, A2220) was added into the samples and incubated overnight with rotation at 4°C. Samples were centrifuged for 1 minute at 2000 g and supernatant was removed. Samples were washed sequentially with the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.0, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.0, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1 mM EDTA, pH8.0, 1% NP-40, 1% Sodium Deoxycholate, 10 mM Tris-HCl, pH8.0). DNA was eluted with 120 μL elution buffer (1% SDS, 100 mM NaHCO3) by vortexing slowly for 15 minutes at 30°C. Samples were centrifuged for 1 minute at 2000 g and supernatant was transferred into a new tube. Four point eight μL of 5 M NaCl and 2 μL RNase A (10 mg/mL) were added and samples were incubated at 65°C overnight. Two μL proteinase K (20 mg/mL) was added and samples were incubated at 60°C for 1 hour. The DNA was subsequently purified using phenol: chloroform extraction.

Drosophila genetics and infection

Fly stocks are maintained on standard fly food. UAS-shEdis transgenic flies were generated using the Valium 20-shEdis construct that integrated into the attp2 site on the 3^rd chromosome (Bestgene). pUAST-Edis or the control pUAST-Laccase2 MCS exon vector transgenic flies were generated using the corresponding pUAST constructs (Bestgene). To generate Edis knockdown or overexpression flies, we crossed UAS-shEdis or pUAST-Edis transgenic lines with the da-Gal4; tub-Gal80^ts composite line [43]. Control cross was set up using a shRNA transgenic line, which expresses an artificial shGFP RNA, or pUAST-Laccase2 MCS exon vector transgenic flies. To minimize lethality at early developmental stages due to the requirement for Edis in development, fly crosses were kept at permissive temperature (18°C) until adult progeny of the appropriate genotype emerge. Subsequently the progeny was shifted to restrictive temperature (29°C) for 5–7 days to allow for transgene expression. We also crossed the shRNA lines to strong ubiquitously expressed drivers Tubulin-Gal4 and Actin-Gal4, or the intermediate ubiquitously expressed driver da-Gal4, and kept the crosses at various temperatures (18, 22 and 25°C). These crosses lead to complete and near complete lethality, respectively. To knock down or overexpress Edis in different tissues, we crossed the shRNA or pUAST lines to tissue-specific drivers including Elav- and nSyb-Gal4 (neuron), and Repo-Gal4 (glia). In addition, we employed a similar strategy and used the UAS-shEct4 and UAS-dsEct4 lines for depleting the linear Ect4 in various tissues. For fly infection experiments, male progeny of the appropriate genotype were either left untreated (as control) or pricked with a sharp needle previously dipped in a concentrated suspension of either the Gram-negative bacteria E. coli or Gram-positive bacteria M. luteus. Flies were harvested 6 or 24 hours post infection, respectively. Levels of mRNAs encoding the antimicrobial peptide genes such as Diptericin, Drosomcyin, and the control RpL32 (rp49) mRNA were analyzed by RT-qPCR. To measure host survival and pathogen clearance, infection experiments were carried out by injecting 9.2 nL of bacterial suspension or PBS (control) into flies using Nanoject II (Drummond). For fly survival experiment, a concentrated culture of Ecc15 (OD₆₀₀ ~5–10) was injected. Survival (in multiple groups of 20–25 flies per group) was monitored daily post infection. To determine pathogen load, a concentrated culture of Ecc15 (OD₆₀₀ ~5–10) were injected into flies. Subsequently, groups of 4 flies were harvested at various time points post infection and homogenized in 200 μl of sterile PBS. Diluted fly homogenates were plated onto LB plates containing Ampicillin. Colony forming units (CFU) were recorded after 24 hours and CFU per fly is calculated.

Drosophila lifespan and locomotor activity

UAS-shEdis or the control UAS-shGFP lines were crossed with the da-Gal4; tub-Gal80^ts composite line. Fly crosses were kept at permissive temperature (18°C) until adult progeny of the appropriate genotype emerge. Subsequently, the progeny was shifted to restrictive temperature (29°C) and survival of flies (in multiple groups of 20–25) was monitored daily. For neuron-specific knockdown, UAS-shEdis or the control UAS-shGFP lines were crossed with the Elav-Gal4 or nSyb-Gal4 drivers. Fly crosses were kept at 22°C until adult progeny of the appropriate genotype emerge. Subsequently, the progeny was shifted to 25°C and survival of flies (in multiple groups, 20–25 flies per group) was monitored daily. To measure locomotor activity, we followed the protocol reported by Liu et al [75] with minor modifications. Briefly, flies in multiple groups of 15 were placed into conical culture tubes and tapped to the bottom, and the percentage of flies that can climb over the 2-centimeter mark within 15 seconds was recorded.

Immunofluorescence

For mushroom body staining, adult brains were dissected, stained and imaged as described [76]. Briefly, adult fly heads of proper ages and genotypes were dissected in PTN buffer (0.1 M Sodium Phosphate Buffer, pH 7.2, 0.1% Triton X-100) and fixed with 4% paraformaldehyde (Alfa Aesar, 43368) for 20 minutes at room temperature, followed by two times rinsing in PTN buffer and then three times washing with PTN buffer for 20 minutes each time. Samples were blocked with 5% BSA in PTN buffer at room temperature for 30 minutes and incubated with primary antibody at 4°C for 72 hours, followed by extensive washing as indicated above. Afterwards, samples were incubated with secondary antibody at 4°C overnight, followed by same extensive washing steps showing above. Primary antibodies: mouse anti-Fasciclin II (1D4, Developmental Studies Hybridoma Bank, developed by Dr. Corey Goodman) at 1:50 dilution, rat anti-Elav antibody (7E8A10, Developmental Studies Hybridoma Bank, developed by Dr. Gerald M. Rubin) at 1:100 dilution, prepared in 1XPBT with 5% normal donkey serum to block nonspecific binding. Secondary antibodies: AlexaFluor 488 (Invitrogen, A-21202) or 596-conjugated donkey-anti-rat antibody and AlexaFluor596-conjugated donkey anti-mouse antibody (Invitrogen) at 1:1000 dilution in 1XPBT. Sample were counter-stained with DNA dye 4’,6’-diamidino-2-phenylindole (DAPI) at 1:10,000 dilution of 1mg/mL stock solution in 1XPBT solution for 10 minutes. For phalloidin staining of adult Drosophila retinas, the heads were dissected in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and fixed with 4% paraformaldehyde for 1 hour. Subsequently, retinas were dissected and fixed for an additional 30 minutes. Retinas were rinsed three times with PBST (PBS + 0.4% Triton X-100) and incubated with 100 nM phalloidin (Cytoskeleton Inc., 057) overnight at 4°C, followed by three times washing. For giant fiber system of adult brain, the heads were dissected in PBS and fixed with 4% paraformaldehyde for 20 minutes, followed by three times washing with PBS. Then all the prepared samples were mounted in 80% glycerol and imaged using a Zeiss compound fluorescent microscope (Zeiss Axio Imager Z1) and processed by Zen software (Zeiss), or Nikon confocal microscope (Nikon A1, Tokyo, Japan). To visualize synapses, immunocytochemistry of dissected 3^rd instar larvae was performed as described previously [77]. The rabbit anti-DVGlut (Drosophila vesicular glutamate transporter) was described previously [78] and used at 1:5,000 dilution. Goat anti-HRP antibody was obtained from Jackson Immunoresearch and used at 1:1000 dilution (123-165-021).

For immunostaining of cultured cells, S2 cells stably transfected with pMT-Flag-Edis-p or pMT vector were transfected with pMT-Relish-HA. Two days post transfection cells were treated with 200 μM CuSO₄ for 2 days. Cells were washed by PBS and fixed in 4% formaldehyde for 10 min at room temperature. After washing with PBST, cells were blocked with 2% BSA in PBST for 30 min. Cells were subsequently incubated with anti-Flag antibody (1:1000, Sigma, F3165) and anti-HA (1,1000, Cell Signaling Technology, #C29F4) overnight at 4°C, washed and incubated in secondary antibodies (anti-mouse 488, 1:1000; anti-Rabbit 594, 1:1000) for 1 hour in room temperature and washed with PBST. Images were acquired on a Nikon confocal microscope. Fluorescence intensity was analyzed by NIS-Elements Viewer.

Natural infection of larvae and tracheal branch imaging

We followed the protocol of Akhouayri et al with minor modifications [64]. An overnight Ecc15 culture was subjected to centrifugation at 4,000 g for 10 minutes at 4°C. The pellet was resuspended in fresh LB liquid media. Around 200 μL of the resulting cell paste (estimated OD₆₀₀  =  200) was directly added on top of the standard medium containing feeding third instar larvae at 25°C. A similar volume of LB broth was used as a control. After 24 hours, the fed larvae were placed in a drop 100% glycerol on glass microscope slide. The slide was heated at 70°C for 10 seconds, and Drosomycin transcription was monitored by fluorescence analysis using Drs-GFP reporter.

Cell culture, transfection, and RNAi

Drosophila S2 cells are maintained in Schneider’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). S2 cells stably expressing shEdis were generated by transfection with Valium 20-shEdis, pMT-Gal4, and the selection marker plasmid pHS-neo using the calcium phosphate method, followed by selection in medium containing 400 μg/mL G418 (Calbiochem). A control cell line expressing shRNA targeting the w gene serves as control. To overexpress Edis, S2 cells were transfected with Hy_pMT Laccase2-Edis, followed by selection in medium containing 400 μg/mL hygromycin (Calbiochem). Cells stably transfected with Hy_pMT Laccase2 MCS exon vector serve as control. Cells were grown in culture medium containing CuSO₄ (final concentration 250 μM) for ~3 and 5 days, respectively, to achieve Edis overexpression and knockdown. For transient knockdown in S2 cells, dsRNA treatment was performed as described previously [36–38]. Briefly, ~2 × 10⁶ S2 cells were seeded in 6-well plates for 24 hours and then transfected with 3 μg of the appropriate dsRNA using the calcium phosphate protocol. Two days later, the cells were harvested, replated in 6-cm plates for 24 hours, and then transfected again with another 9 μg of dsRNA. Cells were harvested three days after the second dsRNA treatment.

Peptidoglycan, bacterial and ecdysone treatment in S2 cells

To generate samples for RT-qPCR analysis, Drosophila S2 cells were treated with 1 μM 20-hydroxyecdysone (Sigma) for 24 hours. Subsequently, cells were treated with 10 μg/mL crude LPS prep (Sigma), which contains peptidoglycan (PGN) as a potent inducer of IMD signaling [17], for another 3 to 6 hours. To generate samples for RNA-seq analysis, S2 cells (at ~ 2X10⁶ cells/mL) were 1) left untreated, 2) treated with 1 μM 20-hydroxyecdysone (Sigma) for 24 hours, 3) treated with a mixture of overnight cultures of E. coli 1106 and M. luteus for 48 hours, or 4) treated with 1 μM 20-hydroxyecdysone for 24 hours and subsequently with a mixture of overnight cultures of E. coli 1106 and M. luteus for an additional 48 hours. Total RNA was extracted and levels of the antimicrobial peptide genes Diptericin, Attacin A, Metchnikowin and the control RpL32 mRNA were analyzed by RT-qPCR. For Relish processing experiments, cells were first treated with 20-hydroxyecdysone (Sigma) for 24 hours. Subsequently, cells were treated with 10 μg/mL crude LPS prep for 10–15 minutes. Cell lysates were prepared and subjected to immunoblot using anti-Relish antibody. The ratio of cleaved/full length Relish was employed for quantification of Relish processing. For Edis overexpression and knockdown experiments, the corresponding stable cells were first treated with CuSO₄ (250 μM) for ~3 and 5 days, respectively, to achieve Edis overexpression and knockdown, prior to 20-hydroxyecdysone and PGN treatment.

In vitro Relish processing

Purification of recombinant proteins was processed as previously described [72]. Briefly, 50 mL of stably transfected cells (Flag-Dredd, Flag-Ran or Flag-Edis) were plated at a density of 10⁶ cells/mL. After 24 hours, cells were treated with 500 μM copper sulfate for 24 hours and cells were collected and lysed in 1.5 mL lysis buffer. The supernatants were incubated with 100 μL anti-Flag-agarose beads (Sigma) overnight and then the beads were eluted with 300 μL 3XFlag-Peptide (100 μg/mL; sigma). The eluates were passed through a 10-kDa cut-off filter (Millipore). For in vitro caspase assays, S2 cells were lysed in 100 μL lysis buffer per 5 mL of saturated cell culture. Twenty microliters of cell lysate was incubated with various combinations of 10 uL purified Flag-Edis (70 ng), Flag-Ran (80 ng), 10 uL Flag-Dredd (30 ng) and 20 μL 3Xcaspase buffer in a 60 μL reaction system at 30 °C for 2h. The reactions were subsequently subjected to immunoblot analysis using anti-Relish and anti-Flag antibodies.

Protein immunoprecipitation and immunoblotting

Cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM DTT, 1 mM orthovanadate) supplemented with protease inhibitor cocktail (Roche). Cleared total lysates were immunoprecipitated with antibodies against Flag (Sigma F3165). Both input and immunoprecipitated samples were analyzed by SDS-PAGE followed by immunoblotting with antibodies against Flag (Sigma, F7425, 1:5000) and Relish (DSHB, 21F3, 1:1000).

Reporter assay

Briefly, ~5×10⁵ of afore-mentioned S2 stable cells were seeded in 24-well plates the day before transfection. Subsequently, 500 ng att-luc reporter construct (firefly luciferase reporter gene driven by the promoter of the antimicrobial peptide gene Attacin) and 20 ng of actin-Renilla luciferase were transfected into these cells. Two days post transfection, cells were treated with 250 μM copper for ~3 and 5 days, respectively, to achieve Edis overexpression and knockdown. Next, cells were treated with 1 μM 20-hydroxyecdysone (Sigma) for 24 hours and subsequently left untreated or treated with PGN for 6 hours. Cell suspensions were arrayed in 384-well plates and reporter activity was measured using the Dual-Glo system (Promega). For data processing, firefly/Renilla ratio was calculated and normalized against the control sample.

RNase R treatment and RT-qPCR

Two protocols were used. 1) We followed the protocol of Salzman et al. Total RNA was isolated with TRIzol, either treated with RNase R at 37°C for 1 hour or mock treated. The RNA samples were subsequently reverse transcribed using Superscript III (Invitrogen) and random hexamer primers, and levels of circular and linear RNAs were measured by quantitative PCR. Levels of circular and linear RNAs prior to and after RNase R treatment were compared. 2) We followed the protocol of Rybak-Wolf et al with minor modifications: Total RNA was treated with 3U/μg RNase R (RNR07250, Lucigen) for 15 minutes at 37°C or mock treated. The RNA was immediately transferred to ice, spiked with 10% total RNA from mouse brain. Subsequently, the RNA samples were subjected to acidic phenol extraction, ethanol precipitation, and reverse transcription. Levels of circular and linear RNAs were quantified by quantitative PCR. Levels of mouse gapdh transcript were employed for normalization. Quantitative PCR was performed using the iQ SYBR-green reagents on a CFX96 Real-Time PCR Detection System (Bio-Rad). Fold changes in RNA levels were calculated using the ΔΔCt method.

Circular RNA identification and RNA-seq Data Analysis

Raw reads (fastq files) were pre-processed by trimming the adapter sequences and filtering out the low-quality reads (reads with base quality < = 20). The detection of circular RNA was based on the computational pipeline described in Memczak et al [46]. The raw reads (paired-end, 2x100) were mapped to Drosophila reference genome (dm3) using bowtie2 [79]. The find_circ program with the same parameters recommended by developer was applied for detecting circRNAs using those unmapped reads [46].