A dynamic Dif/lncRNA-CR42715/miR-965-3p feedback loop orchestrates toll pathway immunity response in Drosophila
Xiaolong Yao, Lin Zhou, Rui Wu, Guorong Yang, Ning Li, Shengjie Li, Fei Ma, Ping Jin
TL;DR
The study reveals a feedback loop involving a transcription factor, a long non-coding RNA, and a microRNA that controls immune responses in fruit flies.
Contribution
A novel TF-lncRNA-miRNA feedback loop regulating Drosophila Toll pathway immunity is identified.
Findings
Dif activates CR42715, which sponges miR-965-3p to enhance Dif protein synthesis and Toll signaling.
Disruption of the CR42715/miR-965-3p feedback loop leads to dysregulated immune responses and reduced host survival.
CR42715 acts early to boost immunity, while miR-965-3p later dampens the response, ensuring a self-limiting immune reaction.
Abstract
The innate immune response requires precise spatiotemporal regulation for organisms to ensure effective pathogen clearance while avoiding detrimental overactivation. Although the core components of the Drosophila Toll pathway are well-established, the post-transcriptional regulatory networks, particularly those involving non-coding RNAs (ncRNAs), remain incompletely understood to date. Here, we elucidate a novel tripartite feedback loop comprising the long noncoding RNA (lncRNA) CR42715, the microRNA (miRNA) miR-965-3p, and the transcription factor (TF) Dif that dynamically modulates Drosophila Toll signaling. Firstly, our results demonstrate that upon Gram-positive bacterial challenge, Dif activates CR42715 expression, which acts as a competitive endogenous RNA (ceRNA) by sponging miR-965-3p to alleviate miR-965-3p-mediated repression of Dif and enhance Dif protein synthesis, thus…
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Fig 8- —http://dx.doi.org/10.13039/501100001809National Natural Science Foundation of China
- —http://dx.doi.org/10.13039/501100004608Natural Science Foundation of Jiangsu Province
- —http://dx.doi.org/10.13039/501100004608Natural Science Foundation of Jiangsu Province
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Taxonomy
TopicsInvertebrate Immune Response Mechanisms · Aquaculture disease management and microbiota · Insect symbiosis and bacterial influences
Introduction
Drosophila melanogaster is a powerful and genetically model organism for studying innate immune responses that are conserved across metazoans, including humans [1,2]. The Drosophila innate immune system mainly comprises cellular immunity and humoral immunity. The activation of cellular immune response includes recognition, phagocytosis, encapsulation and killing parasites [3]. The humoral immune response is mainly based on the recognition of microorganisms by pattern recognition receptors leading to the production of antimicrobial peptides (AMPs), such as Drosomycin (Drs), Metchnikowin (Mtk) and others [4–7]. The humoral immune response is centrally mediated by two primary signaling cascades, the Toll and the Immune deficiency (Imd) pathways [8,9]. The Toll signaling pathway is essential for Gram-positive bacteria and fungi [8,10]. Toll signaling is activated when the ligand Spaetzle binds the Toll ectodomain [11]. Upon activation, Toll transmits signals via the adaptor protein Myd88 and the kinase Pelle (Pll), leading to the phosphorylation and subsequent degradation of Cactus (Cact). Cact is a cytoplasmic protein containing ankyrin repeats that typically suppresses the activity of NF-κB family transcription factors Dorsal (Dl) and Dorsal-related immune factor (Dif) by retaining them within the cytoplasm [12,13]. Dif and Dorsal share structural similarity and both can translocate into the nucleus to regulate the expression of AMP genes within the Toll signaling mediated innate immune response. Previous genetic study has revealed their distinct functional specializations that Dif is the primary factor required for the induction of the key AMP Drs following immune challenge, while Dorsal appears to play a limited role in this immune response [14]. This specific requirement for Dif in a major arm of the Drosophila immunity makes it a focal point for understanding the precise regulatory role of Toll signaling to ensure an effective and balanced defense against infection.
Immune overactivation or deficiency can cause host damage and reduce viability. Therefore, the intensity and duration of the immune response in Drosophila must be finely regulated to maintain immune homeostasis and ensure normal viability. Notably, several protein-coding genes have been reported to activate the Toll signaling pathway, including the E3 ubiquitin ligase Sherpa [15], Mop and Hrs [16], Dicer-2 [17], Gprk2 [18] and Cactin [19]. In contrast, negative regulators such as Senju [20], Pellino [21], Krz and Ulp1 [22], Ubc9 [23] and Cactus [24] have been identified to suppress Toll signaling in Drosophila. Beyond the protein-based regulatory network, non-coding RNAs contribute significantly to Drosophila innate immunity, among which miRNAs have been most notably documented for their post-transcriptional regulation. In the context of the Drosophila Toll pathway, miR-8 maintains immune homeostasis of the Toll pathway by directly targeting both Toll and Dorsal [25]. Similarly, the miR-958 inhibits Toll, and miR-310 family suppresses Drs expression [26,27]. In particular, miR-317 targets isoform-specific transcript of Dif, collectively contributing to the negative regulation of Toll signaling [28]. However, the role of lncRNAs in regulating the Drosophila innate immune pathway remains relatively understudied.
LncRNAs, defined as RNA transcripts longer than 200 nucleotides do not encode proteins, exhibit even greater functional diversity as signals, decoys, guides or scaffolds to regulate gene expression at the epigenetic, transcriptional, and post-transcriptional levels [29,30]. Although the regulatory roles of lncRNAs are well-established in mammalian innate immunity, they remain poorly characterized in Drosophila. Pioneering work identified lncRNA-VINR as the first Drosophila lncRNA involved in immunity, demonstrating its participation in antiviral responses and regulation of the Toll pathway [31]. Recently, lncRNA-CR46018 and lncRNA-CR33942 have been reported to enhance AMP expression by interacting with the TF Dif/Dorsal and promoting their binding to the promoters of target AMP genes in the early phase of Gram-positive bacterial infection [32,33]. Conversely, during the mid-to-late stages of Micrococcus luteus (M.luteus) infection, lncRNA-CR11538 acts as a protein decoy by binding to Dif/Dorsal, thereby preventing them from binding to AMP promoters [34]. This mechanism suppresses AMP expression and prevents excessive immune activation, facilitating the restoration of immune homeostasis. Although these findings establish a complex and dynamic regulatory landscape where protein-coding genes and ncRNAs interact to orchestrate a precise and balanced immune defense, more lncRNAs involved in fine-tuning Drosophila Toll pathway and the underlying mechanisms governing these immune-regulatory lncRNAs themselves remain poorly understood.
While the individual roles of specific lncRNAs or miRNAs in modulating the Toll signaling pathway have become increasingly clarified, a critical knowledge gap persists regarding potential crosstalk and hierarchical regulatory interactions among TF, lncRNA and miRNA during Drosophila innate immunity. Here, we uncover a novel regulatory mechanism whereby lncRNA-CR42715 inhibits miR-965-3p expression by sponging this miRNA, which is known to act as a negative modulator of the Toll pathway during Gram-positive (G⁺) bacterial infection. Interestingly, this proposed regulatory cascade culminates in a feedback loop: Dif transcriptionally activates CR42715, thereby establishing a sophisticated, previously uncharacterized network for precise fine-tuning of Toll pathway activity. Therefore, elucidating this Dif/CR42715/miR-965-3p feedback loop is essential for advancing our comprehensive understanding of how immune responses are initiated, sustained, and ultimately resolved to maintain organismal homeostasis.
Results
lncRNA-CR42715 is involved in Drosophila toll pathway immune response and lacks protein-coding capability
To identify novel lncRNAs that may regulate the Toll signaling pathway in Drosophila, we analyzed previously published RNA‑seq data from flies infected with M.luteus [35]. The expression of CR42715 exhibited significant changes upon immune stimulation. To further validate its immune relevance, we examined CR42715 expression in w^1118^ flies at different time points (3, 6, 12, 24, 48 h) after infection with M.luteus or PBS (Fig 1A). Compared with the control group, CR42715 expression was significantly higher in M.luteus-stimulated flies at all time points, peaking at 6 h and then declining by 12 h. These findings suggest that CR42715 may be involved in the Drosophila Toll pathway immune response following Gram-positive bacterial infection.
*CR42715 is a real lncRNA associated with immunity.(A) The dynamic expression patterns of CR42715 in the wild-type flies after M.luteus or PBS infection at different time points (0 h, 3 h, 6 h, 12 h, 24 h and 48 h). (B) Coding potential prediction of CR42715 using the CPC2 (Coding Potential Calculator 2) algorithm. Dif is coding RNA used as negative controls. CR43306-RA and CR43306-RB are lncRNAs used as positive controls. (C) An in vitro transcription/translation assay showed that CR42715 has no detectable polypeptide products. CR42715: full-length; CR42715 Antisense: antisense of full-length CR42715; Positive control: Luciferase gene driven by the T7 promoter used as a positive control. (D) qRT-PCR analysis of CR42715 expression at the nuclear and cytoplasmic levels in S2 cells. U6 was used as a nuclear marker, and GAPDH was used as a cytosolic marker. Data are presented as mean ± SEM from three independent biological experiments. Statistical significance was determined by Student’s t-test, with *P < 0.01 and ns indicating no significant difference.
In this study, we further employed both in silico and experimental approaches to assess the coding potential of CR42715. CPC2 tool analysis revealed that, compared with the protein Dif, both CR42715 and its antisense transcript possess extremely low protein-coding potential. This feature is consistent with that of known non-coding RNAs, such as the well-characterized lncRNA CR43306 [36,37] (Fig 1B). In vitro expression experiments also indicated that CR42715 lacks protein-coding capacity (Fig 1C). Additionally, because the functional mechanisms of lncRNAs are closely associated with their subcellular localization [38], we analyzed the distribution of lncRNA-CR42715 in Drosophila S2 cells. RT-qPCR analysis of nuclear and cytoplasmic fractions revealed that approximately 80% of CR42715 is localized in the cytoplasm (Fig 1D). Together, this study identifies lncRNA-CR42715 as a novel immune-responsive factor in Drosophila Toll pathway that functions independently of protein-coding ability.
CR42715 promotes AMP expression and enhances host survival
To investigate the potential regulatory role of lncRNA-CR42715 in immune responses, we generated CR42715 overexpression (CR42715-OE) and CR42715 RNAi (CR42715-KD) strains (S1A and S1B Fig). We then infected CR42715-OE flies (Gal80^ts^; Tub-Gal4 > CR42715) and control flies with Micrococcus luteus (M.luteus) and measured the transcript levels of two hallmark Toll pathway effector AMPs, Drosomycin (Drs) and Metchnikowin (Mtk), which serve as canonical readouts of Toll pathway activity. In CR42715-OE flies, Drs expression was significantly upregulated at 3, 6, and 24 h post-infection, while Mtk expression was markedly increased at 12 and 24 h (Fig 2A-2B). Conversely, in CR42715-KD flies, both Drs and Mtk levels were significantly reduced at 6 and 12 h after infection (Fig 2C-2D).
*CR42715 positively regulates the immune response of the Drosophila Toll pathway.(A-B) The expression levels of Drs and Mtk in CR42715 overexpressed (Gal80ts; Tub-Gal4 > CR42715) and control flies at different time points (0 h, 3 h, 6 h, 12 h, 24 h) after M.luteus stimulation. (C-D) The expression levels of Drs and Mtk in CR42715 RNAi (Gal80ts; Tub-Gal4 > CR42715 RNAi) and control flies at different time points (0 h, 3 h, 6 h, 12 h, 24 h) after M.luteus stimulation. (E) The protein levels of Drs-GFP were detected in control flies, CR42715 overexpressed and CR42715 RNAi flies with M.luteus stimulated at 6 h. (F) ImageJ was used for fluorescence quantitative analysis and statistical analysis. (G) PBS and E. faecalis were injected to observe the changes in the survival rate of flies within 7 days. Gal80ts; Tub-Gal4/ + flies were used as a control. Gal80ts; Tub-Gal4/ + flies (n = 50); Gal80ts; Tub-Gal4 > UAS-CR42715 flies (n = 50); Gal80ts; Tub-Gal4 > UAS-CR42715 RNAi (n = 50). For each phenotype, 50 flies per group were included for recording survival. The log-rank test (GraphPad Prism 6.0 software) was used to calculate the statistical significance of the fruit fly survival experiment. The experiments were performed for three independent biological replicates with three technical replicates for each experiment (five flies pooled in each biological replicate per genotype). The bars represent the mean ± SEM. *p < 0.05, **p < 0.01, **p < 0.001 (unpaired two-tailed Student t te sts).
To further assess the effect of CR42715 on Drs at the protein level, we employed a Drosomycin promoter-driven GFP reporter (Drs-GFP) in the background of CR42715-OE, CR42715-KD, and control flies. Consistent with the above transcriptional data, GFP fluorescence intensity was significantly enhanced in CR42715-OE flies and reduced in CR42715-KD flies at 6 h post-infection with M.luteus (Fig 2E-2F). Together, these results indicate that lncRNA-CR42715 acts as a positive regulator of the Drosophila Toll signaling pathway.
To evaluate the physiological relevance of CR42715 in innate immunity, we monitored the survival of CR42715-OE, CR42715-KD, and control flies following infection with the lethal Gram-positive bacterium Enterococcus faecalis (E. faecalis). CR42715-OE flies displayed a significantly higher survival rate compared to controls, whereas CR42715-KD flies showed markedly reduced survival (Fig 2G). Collectively, these findings demonstrate that CR42715 not only modulates the expression of key Toll pathway effectors but also actively influences host survival, supporting its role as a functional positive regulator of the Drosophila immune response.
CR42715 acts as a molecular sponge for miR-965-3p to derepress Dif and potentiate toll signaling
The above result reveals that CR42715 is predominantly localized in the cytoplasm, which suggests that CR42715 may participate in immune regulation through a ceRNA mechanism. To identify candidate miRNAs that interact with CR42715, we employed TargetScan, miRanda, and RNA22 for in silico prediction, and intersected the results with our previously generated Drosophila Toll pathway-related small RNA-seq datasets [35]. This integrated approach identified three candidate miRNAs (miR-311-3p, miR-34-3p, and miR-965-3p) associated with the Toll pathway (S2A Fig). Subsequent quantitative analysis demonstrated that CR42715-OE led to reduced expression levels of miR-965-3p and miR-34-3p, but did not affect miR-311-3p (S2B Fig). Notably, bioinformatic predictions suggested that miR-965-3p, but not miR-34-3p, may target Dif, a key transcription factor in the Toll signaling pathway. We therefore focused subsequent investigations on validating the functional relationship between CR42715 and miR-965-3p.
First, we confirmed that miR-965-3p express decreased in CR42715-OE flies and increased in CR42715- KD flies (Fig 3A), indicating a genetic interaction between CR42715 and miR-965-3p. To assess direct binding, we performed a luciferase reporter assay in Drosophila S2 cells, finding that miR-965-3p significantly suppressed luciferase activity of the reporter construct containing the wild-type target sequence (luc-CR42715), whereas mutation of the predicted binding site (luc-CR42715 MUT) abolished this repression (Fig 3B-3C). Furthermore, RNA immunoprecipitation (RIP) assays confirmed the endogenous association of both CR42715 and miR-965-3p with Ago1-centered RNA-induced silencing complex (RISC), as indicated by their significant enrichment in anti-Ago1 immunoprecipitates compared to the IgG control (Fig 3D-3E). Collectively, these results demonstrate that CR42715 can act as a molecular sponge to inhibit the activity of miR-965-3p.
*Verification of the miRNA directly targeted by CR42715.(A) The expression of miR-965-3p in CR42715 overexpressed, CR42715 RNAi and control flies. (B) The binding site of miR-965-3p in CR42715 transcript. (C) Luciferase reporter assay showed that over-expression of miR-965 significantly reduced the luciferase activity in cells that were transfected with the wild-type CR42715 vector, without reducing the luciferase activity in cells that were transfected with the mutant-type vector. (D-E) Anti‐Ago1 RIP assay verified the combination between miR‐965 and lncRNA–CR42715. (F-G) The expression level of Dif in CR42715 overexpressed flies, CR42715 RNAi flies and control flies at 0 h, 3 h, 6 h, 12 h, 24 h after M.luteus infection. The experiments were performed for three independent biological replicates with three technical replicates for each experiment (five flies pooled in each biological replicate per genotype). The bars represent the mean ± SEM. *p < 0.05, **p < 0.01, **p < 0.001 (unpaired two-tailed Student t tests).
We next examined the expression of Dif, a putative target of miR-965-3p. Upon M.luteus infection, Dif expression in control flies showed a slight upregulation at 6–12 h post-infection. Correspondingly, Dif expression was upregulated in CR42715-OE flies at 3, 12, and 24 h post-infection, whereas it was downregulated in CR42715-KD flies at 6, 12, and 24 h post-infection (Fig 3F-3G). Taken together, our findings support a model in which CR42715 can suppress miR-965-3p, thereby relieving its repression on Dif and enhancing the Toll signaling-mediated immune response.
miR-965-3p negatively regulates Toll signaling by directly targeting Dif
Having established CR42715 as a negative regulator of miR-965-3p, we subsequently investigated whether and how miR-965-3p influences the Toll signaling pathway. We first quantified the expression levels of AMPs in the miR-965-OE (Gal80^ts^; Tub-Gal4 > miR-965) flies and miR-965 KO/ + flies after M.luteus infection (S3A-S3B Fig). The results revealed a significant downregulation of both Drs and Mtk in miR-965-OE flies post-infection (Fig 4A-4B), whereas their expression was elevated in miR-965 KO/ + flies (Fig 4C-4D). To further evaluate the physiological relevance of miR-965 in the immune response, we compared the survival rates of miR-965-OE and miR-965 KO/ + flies after infection with the lethal E. faecalis. Consistent with the AMP expression data, miR-965 overexpression reduced survival, while miR-965 KO/ + enhanced survival relative to the controls (Fig 4E-4F). These findings indicate that miR-965-3p acts as a negative regulator of the Toll pathway in Drosophila.
*miR-965 negatively regulates Drosophila Toll pathway immune response.(A-B) The expression level of Drs and Mtk in miR-965 overexpressed (Gal80ts;Tub-Gal4 > UAS-miR-965) and control flies at 0 h, 12 h, 24 h injected with M.luteus. (C-D) The expression level of Drs and Mtk in miR-965KO/+ and control flies at 0 h, 12 h, 24 h injected with M.luteus. (E) The variation of the survival rate was observed in miR-965 overexpressed flies, and control flies with PBS as well as E. faecalis infection. Tub-Gal4 > /+ (n = 50); Tub-Gal4 > UAS-miR-965 (n = 50). The number of flies injected with PBS group or E. faecalis group was consistent. (F) The survival rates of the miR-965KO/+ and control flies w1118 were monitored after PBS and E. faecalis infection. w1118 (n = 50); miR-965KO/+ (n = 50). The number of flies injected by PBS group or E. faecalis group was consistent. (G-H) The expression level of Dif in miR-965 overexpressed, miR-965KO/ + flies and control flies at 0 h, 12 h, 24 h after M.luteus infection. (I) Potential binding site of miR-965 in Dif CDS and Dif CDS mutant. (J) The luciferase activities of the report plasmids without or with mutation sites were detected in Drosophila S2 cells. The experiments were performed for three independent biological replicates with three technical replicates for each experiment (five flies pooled in each biological replicate per genotype). Statistical fly survival analysis was performed using log-rank (Mantel-Cox) tests. The bars represent the mean ± SEM. *p < 0.05, **p < 0.01, **p < 0.001 (unpaired two-tailed Student t tests).
We further investigated whether miR-965-3p represses its putative target gene Dif. In miR-965-OE flies, Dif expression was significantly reduced at both 12 and 24 h after M.luteus infection. In contrast, Dif levels were markedly increased at 24 h in miR-965 KO/ + flies (Fig 4G-4H). Using a luciferase reporter assay in Drosophila S2 cells, we confirmed that miR-965-3p directly binds to a coding sequence (CDS) region of Dif mRNA, rather than the canonical 3’UTR or Dif-CDS MUT sequence (Fig 4I-4J).
In summary, our results demonstrate that miR-965-3p directly targets the CDS of Dif mRNA to attenuate Toll signaling. This repression is itself modulated by CR42715, to further clarify the regulatory relationship among CR42715, miR-965-3p, and Dif, we performed an additional dual-luciferase reporter assay to examine their combined effects on Dif (S3C Fig). The results provide direct functional evidence that CR42715 counteracts miR-965 activity and regulates Dif expression, thereby further supporting the proposed CR42715-miR-965-Dif regulatory axis.
Dif transcriptionally activates CR42715 to establish a positive-feedback loop
Although our above findings have revealed a linear regulatory axis in which CR42715 enhances the immune response by acting as a ceRNA that sequesters miR-965-3p, which in turn suppresses Dif, the regulatory mechanism controlling CR42715 expression during immune activation remained unclear. Dif, a key transcription factor in the Toll signaling pathway, is known to directly activate the expression of some AMPs and other immune-related genes [26,27]. To investigate whether Dif also regulates CR42715, we analyzed publicly available Dif ChIP-seq data from the ENCODE database and visualized using IGV 2.6.2. Using the FIMO tool from the MEME suite, we identified significant Dif binding peaks within the upstream promoter region of CR42715 (S4A-S4B Fig). We then performed ChIP-qPCR with primers targeting this putative promoter region and observed pronounced enrichment of Dif-Flag at the CR42715 promoter (Fig 5A), confirming this binding in vivo. Moreover, dual-luciferase reporter assays in S2 cells co-transfected with a Dif expression plasmid showed strong activation of the wild-type CR42715 promoter, but not a mutated version (Fig 5B), further supporting direct transcriptional regulation by Dif. We also examined CR42715 expression in Dif-overexpression (Dif-OE) and Dif-RNAi (Dif-KD) Drosophila lines (Fig 5C) and found that CR42715 levels were significantly increased and decreased, respectively, compared to controls (Fig 5D). Together, these results demonstrate that Dif directly binds to the CR42715 promoter and activates its transcription. Thus, the lncRNA-CR42715/miR-965-3p/Dif axis not only releases Dif from miR-965-3p–mediated repression but also establishes a positive feedback loop that amplifies the Drosophila Toll pathway immune response.
*Dif directly promotes the transcription of CR42715.(A) ChIP-qPCR was performed on CR42715 promoter in S2 cells using anti Dif-flag with Drs promoter as positive control. (B) The reporter activity was determined, together with or without exogenous Dif in Drosophila S2 cell on luciferase assay. (C) The expression level of Dif in Dif overexpressed flies (Gal80ts; Tub-Gal4 > UAS-Dif) and Dif RNAi flies (Gal80ts; Tub-Gal4 > UAS-Dif RNAi). (D) The expression level of CR42715 in Dif overexpressed flies and Dif RNAi flies. The experiments were performed for three independent biological replicates with three technical replicates for each experiment (five flies pooled in each biological replicate per genotype). The bars represent the mean ± SEM. *p < 0.05, **p < 0.01, **p < 0.001 (unpaired two-tailed Student t tests).
The dynamic Dif/CR42715/miR-965-3p feedback loop fine-tunes toll pathway signaling
The discovery of the Dif/CR42715/miR-965-3p feedback loop led us to speculate that it may function as a molecular rheostat, ensuring precise control over the amplitude and duration of Toll pathway output. To test this model, we generated UAS-Toll 10b and UAS-CR42715 RNAi co-overexpression flies (Toll10b + CR42715-KD). The UAS-Toll 10b is a constitutively active Toll receptor variant that sustains high Dif level. As shown in Fig 6A, CR42715 expression was strongly induced (over 3-fold) in Toll 10b flies, but was significantly reduced in Toll 10b + CR42715-KD flies. Consistent with this, miR-965-3p levels were suppressed in Toll 10b flies but restored upon Toll 10b + CR42715-KD strains at 12 h post M.luteus infection (Fig 6B). Consequently, the elevated expression of Dif, Drs, and Mtk observed in Toll 10b flies was partially rescued in Toll 10b + CR42715-KD flies at 12 h post-infection (Fig 6C-6E). Together, these findings support the role of the Dif/CR42715/miR-965-3p feedback loop in amplifying and fine-tuning Toll-mediated immune responses.
*Dif strengthen the Drosophila Toll immune pathway via activating CR42715 transcription.The expression levels of CR42715 (A), miR-965-3p (B), Dif (C), Drs (D) and Mtk (E) in Toll 10b overexpressed flies (Gal80ts; Tub-Gal4 > Toll 10b), Toll 10b and CR42715 RNAi co-overexpression flies and control flies (Gal80ts; Tub-Gal4 > Toll 10b + CR42715 RNAi) were detected at 12 h after M.luteus stimulation. The experiments were performed for three independent biological replicates with three technical replicates for each experiment (five flies pooled in each biological replicate per genotype). The bars represent the mean ± SEM. *p < 0.05, **p < 0.01, **p < 0.001 (unpaired two-tailed Student t tests).
To further delineate the physiological relevance of this loop, we analyzed the temporal expression profiles of key immune components in wild-type flies after M.luteus challenge. The AMP genes Drs and Mtk were progressively induced, peaking at 12 h (>25-fold; Fig 7A and 7B). Similarly, Dif was significantly upregulated at 12 h before declining to baseline by 48 h (Fig 7C). Notably, CR42715 expression was rapidly induced by 3 h, peaked at 6 h, and returned to baseline by 12 h, indicating its role in early immune potentiation (Fig 7D). In contrast, miR-965-3p levels remained stable during the first 6 h but increased markedly by 12 h, peaking at 24 h post-infection (Fig 7E). This reciprocal expression pattern suggests that miR-965-3p acts to restrain excessive Toll signaling in later phases, likely through repression of Dif. Collectively, these coordinated dynamics indicate that the Dif/CR42715/miR-965-3p feedback loop can dynamically orchestrate Toll pathway activity in a time-dependent manner to ensure an effective yet self-limited immune response.
*The temporal expression patterns of genes in the wild-type flies after M.luteus infection.The dynamic expression patterns of Drs (A), Mtk (B), Dif (C), CR42715 (D) and miR-965-3p (E) in the wild-type flies after M.luteus infection at different time points (0 h, 3 h, 6 h, 12 h, 24 h and 48 h). The experiments were performed for five independent biological replicates with three technical replicates for each experiment (five flies pooled in each biological replicate per genotype). The bars represent the mean ± SEM. *p < 0.05, **p < 0.01, **p < 0.001 (unpaired two-tailed Student t tests).
Discussion
Insufficient and excessive immune activation can pose a significant threat to organismal fitness. Therefore, the amplitude and duration of immune signaling must be precisely calibrated for immune homeostasis [39]. While multiple regulatory factors involved in Drosophila innate immune pathway have been identified, the interplay between lncRNA, miRNA and TF in maintaining immune homeostasis remains poorly understood. This study addresses this gap by characterizing a sophisticated tripartite feedback loop comprising CR42715, miR-965-3p and the NF-κB transcription factor Dif. This circuit functions as a molecular rheostat to modulate the intensity and temporal dynamics of the Drosophila Toll pathway immune response.
Unlike our previously described nuclear lncRNAs, such as CR46018 and CR33942, which modulate Drosophila Toll pathway immunity by influencing transcription factor binding at AMP promoters [21,22], CR42715 is primarily localized in cytoplasm. This localization suggests a potential role as a competing endogenous RNA (ceRNA), which we demonstrate directly suppress miR-965-3p to promote the immune response. Notably, we found that miR-965-3p targets the coding sequence (CDS) of Dif mRNA rather than its 3’UTR. This contrasts with the late-phase regulator miR-317, which acts on the Dif 3′UTR [17]. miRNA-mediated regulation through CDS regions is less commonly discussed than canonical 3′UTR sites [40], and in many cases has been associated with translational repression [41]. However, accumulating evidence indicates that miRNA binding to coding sequences can also reduce mRNA abundance, depending on the extent of base pairing, cellular context, and the associated effector mechanisms [42]. In our study, the observed reduction of Dif mRNA upon miR-965-3p overexpression suggests that miR-965-3p/Dif interaction promotes mRNA degradation, however, we could not exclude the possibility that translational repression may also occur. These results indicate that during the immune response, different miRNAs, such as miR-317 and miR-965, may collectively regulate the expression of the transcription factor Dif to achieve effective control of immune intensity.
The core innovation of our work lies in the hierarchical architecture of this regulatory circuit. Dif transcriptionally activates CR42715 in a Toll-dependent manner, while CR42715 post-transcriptionally relieves Dif repression by sequestering miR-965-3p. This creates a coherent feed-forward loop that amplifies the initial immune signal. Crucially, this configuration is transformed into a self-regulating feedback circuit because miR-965-3p represses its own transcriptional inducer (Dif). We propose that this three-layer architecture including transcriptional activation (Dif → CR42715), post-transcriptional sequestration (CR42715-miR-965), and CDS-level repression (miR-965-Dif) constitutes a highly responsive network ideal for rapid, precise immune signal modulation.
The expression dynamics of specific genes following infection (Fig 7) support a model where this feedback loop acts as a biological “rheostat,” enabling finely tuned amplification of the Dif-driven response during the early activation phase (Fig 8). Upon infection, activated Dif protein translocates into the nucleus and triggers the expression of the lncRNA CR42715 as well as antimicrobial peptides. CR42715 then sequesters miR-965 in the cytoplasm, which alleviates miR-965-mediated degradation of Dif mRNA and amplifies the immune response through a positive feedback loop. Later, as Toll signaling and Dif nuclear localization decline, reduced CR42715 levels would allow releasing miR-965-3p to suppress Dif, thus facilitating the attenuation of Toll signaling and the return to immune homeostasis (Fig 8). This temporal dynamic suggests the feedback regulation loop is optimized for initiating a robust yet self-limiting response, a crucial feature for managing infection without excessive or insufficient immune activation. The opposing survival outcomes of CR42715 overexpression and knockdown further underscore the physiological importance of its precise regulation within this loop (Fig 2G). We propose that the selection of CR42715 or miR-965-3p as dominant regulators at different stages is determined by their distinct regulatory properties, with lncRNA regulation enabling rapid and signal-dependent responses, and miRNA regulation providing a more stable and delayed mechanism for fine-tuning and termination of immune signaling. Collectively, these findings indicate that this feedback structure should be particularly poised for the dynamic spatiotemporal control of Drosophila immune responses.
The Molecular mechanism of Dif/CR42715/miR-965 axis promoting immunity in Drosophila.Upon infection, activated Dif protein translocates into the nucleus and triggers the expression of antimicrobial peptides and the lncRNA CR42715. CR42715 then sequesters miR-965 in the cytoplasm, which alleviates miR-965-mediated degradation of Dif mRNA and amplifies the immune response through a positive feedback loop. Later, declining CR42715 levels allow miR-965 to repress Dif expression, thereby restoring immune homeostasis.
In summary, we have delineated a dynamic feedback loop centered on the Dif/CR42715/miR-965-3p axis that extends beyond a simple linear pathway. It embodies a tunable system designed for precise temporal control of the NF-κB response, providing a novel mechanism for achieving immune homeostasis in Drosophila. This work provides a framework for understanding how multi-tiered post-transcriptional networks contribute to the precision regulation of innate immunity.
Materials and methods
Fly culture, stocks, and infections
Flies were maintained at 25°C and 60% relative humidity on a diet containing 10% (w/v) Saccharomyces cerevisiae, 8% fructose, 2% polenta, and 0.8% agar. The w^1118^ strain was used as the wild-type control throughout the study. The fly strains used in this study are provided in the supplementary information (S1 Table). In order to avoid the potential effects of gene overexpression and knockout on Drosophila growth and development, the hybridized fruit flies were cultured in an 18°C incubator. After the larvae emerged as adults and sufficient flies were collected, they were subjected to heat shock at 29°C for 2–3 days. Adult male flies were injected with 15 nl of bacterial suspension or PBS control as previously described (70). These infected flies (five flies per group) were harvested at the designated time point and ground in Trizol reagent (Vazyme, China) for RNA extraction and subsequent experiments. All experiments were performed with three biological replicates. Survival assays were performed by injecting flies with a lethal Gram-positive bacterium, Enterococcus faecalis, and mortality was recorded at 24-hour intervals. The survival analysis was repeated three times, with a minimum cohort size of 50 flies per group.
Bioinformatics prediction
All gene sequences were obtained from Flybase (http://flybase.org/). The binding sites between lncRNA-CR42715 and miRNA were predicted using TargetScan, miRanda, and RNA22 database (https://cm.jefferson.edu/rna22/). Similar methods were also used to predict miRNA binding sites in target genes. The Dif motif sequence on the lncRNA-CR42715 promoter region was predicted via the ENCODE database (https://www.encodeproject.org/) and the FIMO website (https://meme-suite.org/meme/tools/fimo). In silico analysis of the full-length CR42715 sequence reveals CR42715 without any coding probability using the web server for Coding Potential Assessing Tool (CPAT) (http://lilab.research.bcm.edu/).
Nuclear-cytoplasmic fractionation
RNA isolation was carried out following the protocols as previous described [43]. Briefly, a total of 10^7 S2 cells were gently washed with PBS, centrifuged at 1500 rpm, and then resuspended in 5 volumes of lysis buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% [v/v] Nonidet P-40, 1 mM DTT and 1 mM PMSF). Subsequently, the samples were centrifuged at 1000 rpm for 3 minutes to separate the nuclear and cytoplasmic fractions. The supernatants were transferred to fresh tubes and the mixtures were aspirated with filter cartridges to isolate cytoplasmic RNA. Meanwhile, the pellets were treated with ice-cold cell fractionation buffer, and the nuclear RNA was extracted using TRIzol reagent (Vazyme, China). Finally, the extracted RNA was subjected to RT-qPCR analysis. The expression levels of the target genes in the respective fractions were normalized to their input expression levels.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) analysis was carried out using a ChIP kit (Abclonal, China), following the manufacturer’s instructions. Briefly, S2 cells transfected with the pAc-Dif-flag vector were cultured for 48 hours and then incubated at room temperature for 10 minutes to stabilize DNA-protein interactions. Cross-linking was halted by adding glycine (final concentration, 0.125 M). Cell extracts were sonicated with a SCIENTZ-IID machine (China) at 20% power output for 10 minutes to shear DNA into fragments approximately 200–500 bp in size. Extracts from 10^7 cells were incubated overnight with antibodies against Flag (Bioworld, BS67268), while IgG (Bioworld, B00051) antibodies were used as a negative control. Five percent of the extract volume was removed before immunoprecipitation and served as an input control. Subsequently, the extracts were incubated with magnetic beads (RM02915, Abclonal) at 4°C for 3 hours and were washed following the manufacturer’s protocol. DNA fragments from the immunoprecipitated complexes and input controls were released by heating at 65°C overnight and purified using the PCR purification kit (UElandy, China). Purified immunoprecipitated DNA and input DNA were then analyzed by PCR. The list of primers used in this study can be found in S2 Table.
RNA immunoprecipitation (RIP)
Magna RIP RNA-Binding Protein Immunoprecipitation Kit (BersinBio, China) was used in this assay following the manufacturer’s specifications. Briefly, S2 cells were fixed with formaldehyde, lysed using lysis buffer, and sonicated. The supernatant was collected and incubated overnight at 4°C with AGO1 antibodies (abcam, UK) and IgG antibodies (Bioworld, China). Then, the mixture was incubated with magnetic beads (Abclonal, China) at 4°C for 2 hours. The dynabeads were washed according to the kit’s instructions. Finally, RNA was extracted from these complexes and used for qPCR as list in S2 Table.
RNA extraction and RT-qPCR
Total RNA was extracted from flies using the TRIzol reagent (Vazyme, China) following the manufacturer’s instructions. The extracted total RNA was then reverse transcribed into cDNA using HiScript II Q RT SuperMix (Vazyme, China). RT-qPCR was performed using AceQ SYBR Green Master Mix (Vazyme, China). Data analysis was conducted using the relative 2^-△△Ct^ method, with Rp49 and U6 serving as internal reference genes. The primers used in RT-qPCR can be found in S2 Table.
Plasmid construction
The precursor miR-965 hairpin was cloned into the pAc5.1/V5-HisA vector to generate the pAc-miR-965 plasmid. The pAc-luc target sites were constructed by separately inserting lncRNA-CR42715 transcript sequence and Dif CDS. The upstream 2-kb promoter sequence of lncRNA-CR42715 was inserted into the pGL3-Basic plasmid. All sequence sites mutation plasmids were generated using an overlapping PCR-directed method. The primers used in the plasmid construct are listed in S2 Table.
Cell culture and luciferase assay
The S2 cells were cultured in Insect culture medium (Yeasen, China) containing 10% fetal bovine serum (Germini) and 1 × Penicillin-Streptomycin Solution (Beyotime, China) at 28°C. S2 cells were seeded in 24-well plates at a density of 1 × 10⁴ cells per well (in triplicate) and transiently transfected with the indicated vectors using X-tremeGENE HP transfection reagent (Roche, USA), according to the manufacturer’s instructions and previously established methods. 48 hours after transfection, cells were washed twice with PBS and then collected. Luciferase activity was measured using the Dual Luciferase Assay Kit (Vazyme, China) to detect firefly and Renilla luciferase activities. The presented data represent the mean of three independent experiments.
In vitro transcription/translation
In vitro transcription/translation experiments were performed following the protocols provided by the manufacturers. Briefly, T7 promoter containing DNA sequences was used in a TNT Quick Coupled Transcription/Translation System (Promega, USA). Then, 1 μg DNA template was mixed with 40 μl TNT T7 Quick Master Mix, 1 ul methionine (1 mM), 1 μl Transcend Biotin-Lysyl-tRNA, and nuclease-free water for a final volume of 50 μl per reaction. The reaction tube was incubated at 30°C for 90 min, and 1 μl reaction product was added into diluted 5 × SDS loading sample buffer for immunoblot analysis by detecting the signals using streptavidin-HRP (Beyotime, China).
Statistics
The experimental data were collected from three independent biological replicates and presented as means ± SEM. Two-tailed Student’s t-tests were used to determine significant differences between values under different experimental conditions. Log-rank (Mantel-Cox) tests were used for statistical fly survival analysis. Graphs were generated using GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA, USA). A significance level of p < 0.05 was used. *p < 0.05; **p < 0.01; ***p < 0.001.
Supporting information
S1 TableThe fly strains used in this study.(XLSX)
S2 TablePrimers for qRT-PCR and plasmid construction used in this study.(XLSX)
S1 FigThe expression levels of CR42715 in CR42715 ectopic expression flies.The expression levels of CR42715 in CR42715 overexpressed flies (A) and CR42715 RNAi flies (B) compared with control.(TIF)
S2 FigScreening for microRNAs potentially targeted by CR42715.(A) The Venn diagram exhibited that the immune-related miRNAs interact with CR42715. (B) The expression levels of miR-965-3p, miR-34-3p and miR-311-3p in CR42715 overexpressed flies.(TIF)
S3 FigThe ectopic expression of miR-965-3p and the relationships among CR42715, miR-965 and Dif.(A-B) The expression levels of miR-965-3p in miR-965 overexpressed flies and miR-965KO/ + flies compared with control. (C) Dual-luciferase reporter assays were performed to examine the combined regulatory effects of CR42715 and miR-965 on Dif expression. Reporter vectors containing the wild-type coding sequence of Dif (Dif-CDS) or a mutant CDS lacking the miR-965 binding site (Dif-CDS mut) were co-transfected into cells with empty vectors, CR42715, miR-965, or both as indicated.(TIF)
S4 FigThe binding site of Dif in CR42715 promoter.The ChIP-seq of Dif-GFP was visualized using IGV 2.6.2. The motif of Dif and its binding site in CR42715 promoter.(TIFF)
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