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Drosophila miR-964 modulates Toll signaling pathway in response to bacterial infection
Accepted Manuscript Drosophila miR-964 modulates Toll signaling pathway in response to bacterial infection Shengjie Li, Jiao Xu, Lianjie Sun, Ruimin Li, Ping Jin, Fei Ma PII:
S0145-305X(17)30355-5
DOI:
10.1016/j.dci.2017.08.008
Reference:
DCI 2963
To appear in:
Developmental and Comparative Immunology
Received Date: 6 July 2017 Revised Date:
15 August 2017
Accepted Date: 15 August 2017
Please cite this article as: Li, S., Xu, J., Sun, L., Li, R., Jin, P., Ma, F., Drosophila miR-964 modulates Toll signaling pathway in response to bacterial infection, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.08.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Drosophila miR-964 modulates Toll signaling pathway in response
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to bacterial infection
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Shengjie Li1, Jiao Xu1, Lianjie Sun1, Ruimin Li1, Ping Jin1 and Fei Ma1,*
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1 Laboratory for Comparative Genomics and Bioinformatics & Jiangsu Key Laboratory
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for Biodiversity and Biotechnology, College of Life Science, Nanjing Normal University,
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Nanjing 210046, China
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* To whom correspondence should be addressed. Tel: +86 25 85891852; Fax: +86 25
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85891852; Email: [email protected].
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Abstract
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Recent studies suggest that microRNA (miRNA) plays important roles in the
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control of immune response and tolerance. We previously found that the expression
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level of antimicrobial peptide gene Drosomycin (Drs) is decreased in miR-964
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overexpressing flies. Here, we further verified that miR-964 deficiency leads to
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hyper-activation of Drs. In addition, we employed three widely-used bioinformatic
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algorithms to screen potential miR-964 targets. Finally, we identified that miR-964
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modulates Toll signaling pathway, at least in part, by repressing the expression of Drs.
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Taken together, our study identifies miR-964 as a modulator of Toll signaling and
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enriches the repertoire of immune-modulating miRNAs in Drosophila.
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Key words: Toll signaling, miR-964, Drosomycin, Drosophila melanogaster
Abbreviations
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miRNA: microRNA
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TLRs: Toll-like receptors
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AMPs: antimicrobial peptides
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uc: unchallenged
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Drs: Drosomycin
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Spz: spätzle
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3’UTR: 3’-untranslated region
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M. luteus: Micrococcus luteus
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E. faecalis: Enterococcus faecalis
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1. Introduction
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The innate immune response is the first line of defense against microbial
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infections in both insects and mammals (Tanji and Ip, 2005). The Toll signaling
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pathway plays an important role in the Drosophila immune response, which includes
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the phagocytosis of microbes, the encapsulation and killing of parasites (Hultmark,
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2003). The identification of the Drosophila melanogaster Toll signaling pathway
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cascade and the subsequent characterization of Toll-like receptors (TLRs) have
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reshaped our understanding of the immune system (Valanne et al., 2011). It is
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well-known about the molecular components of the Drosophila Toll signaling
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ACCEPTED MANUSCRIPT pathway and their functions in immune response and tolerance against foreign
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pathogens (De Gregorio et al., 2002, 2001; Lemaitre et al., 1997). To date, the focus
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of most studies has been on protein-coding genes (Ji et al., 2014; Ragab et al., 2011).
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However, the discovery of miRNA has revealed an unexpected layer of genetic
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programs that regulate the Drosophila immune response at posttranscriptional levels
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(Chen et al., 2013).
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MiRNA, a class of small non-coding RNAs (~22 nucleotides), mainly regulates
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expression of specific gene through imperfect base pairing with the 3’-untranslated
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region (3’UTR) of mRNA (Bartel, 2009). MiRNA has widespread effects on gene
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expression by influencing target mRNA stability and/or translational efficiency
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(Brennecke et al., 2005). Emerging evidences indicate that miRNAs play important
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roles in impacting the intensity and/or duration of Drosophila immune signaling
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(Choi and Hyun, 2012; Garbuzov and Tatar, 2010; Lee and Hyun, 2014; Xiong et al.,
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2016). Especially, our recent works have demonstrated that miR-310 family
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participates in the Toll-mediated immune response (Li et al., 2017), and miR-958
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directly targets the Toll and Dif genes to negatively regulate the Toll signaling
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pathway (Li et al., 2016). Therefore, systematic analysis of the innate immune
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response in the model organism Drosophila has provided important insights into the
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mechanisms of immune regulation by miRNAs.
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In the Drosophila Toll signaling pathway, Gram-positive bacterial or fungal
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infection leads to the systemic production of antimicrobial peptides (AMPs)
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(Aggarwal and Silverman, 2008). Especially, the antimicrobial peptide Drosomycin (Drs)
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and Hoffmann, 2007). Our previous study showed that the level of AMP gene Drs is
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decreased in miR-964 overexpressing flies, suggesting that miR-964 might negatively
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regulate Toll signaling response in Drosophila (Li et al., 2017). To further prove our
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above hypothesis, in this study, we detailedly investigated the mechanism of miR-964
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regulating AMP expression in Drosophila. Our results revealed that mir-964 directly
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targets the AMP gene Drs to negatively regulate the Toll signaling immune response
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in Drosophila.
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2. Materials and methods
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2.1
Drosophila culture and stocks
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Drosophila stocks were maintained under standard culture conditions. All stocks
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in this study were obtained from the Bloomington Stock Center. The ubiquitous
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temperature sensitive Gal80ts-Gal4 driver system was used to express miRNAs of
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interest specifically in adult flies. Crosses were performed, and the resulting progeny
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were initially raised at the permissive temperature (18°C) for the inhibitor Gal80 to
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repress Gal4 activity. Following adult eclosion, flies were transferred to 29°C to allow
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activation of Gal4 in the adult stage.
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Infection and survival experiments
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Drosophila adult males, aged 3–4 days, were used for septic injury experiments.
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Control and miRNA-overexpressing flies were infected by Micrococcus luteus (M.
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immune response, to induce the expression of Drs. Septic injury was performed by
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pricking the thorax of the flies with a pulled glass capillary carrying M. luteus using a
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Nanoject apparatus (Nanoliter 2010, WPI) (Neyen et al., 2014). Flies were then
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incubated at 29°C and collected at appropriate time point post-infection. For the
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survival experiment, flies were then infected with Enterococcus faecalis (E. faecalis),
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and survival was monitored for 24 h (Valanne et al., 2010).
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RNA extraction and qRT-PCR
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Total RNA was extracted from control and treated adult flies using Trizol
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(Invitrogen). cDNA was synthesized using PrimeScript RT Master Mix kit (Takara).
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Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara) in triplicate on
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an ABI StepOne plus real-time PCR instrument. SYBR Prime Script miRNA RT-PCR kit
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(Takara) was used for quantitative RT-PCR of miRNAs. rp49 and 5s rRNA genes were
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used as reference controls. All qRT-PCR primer sequences are listed in Supplemental
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Table 1.
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In silico prediction of miRNA targets
Toll signaling pathway related genes that are potential targets of Drosophila
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miRNAs were identified using three different miRNA prediction algorithms, i.e.
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TargetScan, miRanda, and PITA (Huang et al., 2011; Li et al., 2010). TargetScan
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predicts biological targets of miRNAs by searching for the presence of conserved
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Ruby et al., 2007). miRanda determines interactions through free-energy dynamics
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across the entire miRNA : mRNA interaction, with increased weight given to the
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miRNA 5’ seed region (Enright et al., 2003). PITA requires sequence similarity within
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the seed region but also determines the accessibility of the mRNA 3’-UTR based on
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free energy (Kertesz et al., 2007).
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2.5
Plasmid construction and luciferase assay
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A miR-964 expression plasmid was constructed from the empty pAc5.1 vector
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(Promega). The 3’-UTR reporter plasmids for spätzle (Spz) and Drs were generated by
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inserting PCR-amplified 3’-UTR fragments downstream of the luciferase gene in the
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pAc5.1 luciferase vector. Binding-site mutant construct was generated via Fast
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Site-Directed Mutagenesis Kit (TIANGEN). Transient transfections were performed in
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S2 cells using X-treme GENE HP transfection reagent (Roche). For dual-luciferase
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reporter assays, cells were harvested at 48 h post-transfection. Firefly and Renilla
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luciferase activity were measured using the Dual-Luciferase Assay Reporter System
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(Promega). Renilla luciferase was used to normalize the transfection efficiency.
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2.6
Western blotting
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For western blotting, the anti-β-actin primary antibody (Bioworld) was used at a
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1:2,000 dilution with an anti-rabbit HRP-conjugated secondary antibody (Vazyme) at
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1:30,000. The antibody against Drs was generated by immunizing rabbits with the
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Escherichia coli-produced recombinant His6-Drs (LSGRYKGPCAVWDNET).
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2.7
Statistical analysis All experimental data in this study were collected from three biological
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replicates. All statistical analyses were shown as means ± SD. Significant differences
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between two groups were determined by two-tailed Student’s t-test. Statistical
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analysis of fly survival experiments was carried out using the log-rank (Mantel-Cox)
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test. For all tests, a P value < 0.05 was considered statistically significant. ns, not
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significant, *P < 0.05; **P < 0.01; ***P < 0.001.
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3. Results
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3.1 miR-964 is involved in negatively regulating the Drosophila Toll signaling
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pathway
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The expression level of Drs is decreased in miR-964 overexpressing flies in our
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previous work (Li et al., 2017), so we speculate that Toll signaling might be
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moderately regulated by miR-964. To further verify whether miR-964 modulates
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Drosophila immune response, we used septic injury with Gram-positive bacteria (E.
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faecalis) to examine the effect of miR-964 on the fly’s survival (Fig. 1A). The miR-964
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overexpressing flies (Gal80ts; Tub>miR-964), control flies (w1118) and miR-964
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knockout flies (miR-964 KO) were infected by pricking with E. faecalis, respectively.
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The miR-964 overexpressing flies showed a statistically significant reduction in
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survival compared with both the control flies and miR-964 knockout flies. However,
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ACCEPTED MANUSCRIPT there was no difference between the miR-964 knockout flies and control flies. It’s
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worth noting that the miR-964 knockout flies had a more significant effect on fly’s
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survival than control flies by compared with the miR-964 overexpressing flies. These
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findings suggested that miR-964 might influence the host defense ability against
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Gram-positive bacterial infection. Next, temporal expression patterns of miR-964
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were analyzed following exposure to other bacterial (M. luteus) compared with the
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physical injury control (Fig. 1B). Our results revealed that the expression of miR-964
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in flies infected by M. luteus was significantly higher than the injured flies at 24 h, 48
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h and 72 h after challenge, indicating that Drosophila host defense causes the
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fluctuation of expression level of endogenous miR-964. We further infected miR-964
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overexpressing flies and miR-964 knockout flies with M. luteus, and tested the
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expression level of Drs at 0 h, 12 h, 24 h post-infection by qRT-PCR. As expected, the
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Drs expression in miR-964 overexpressing flies (Gal80ts; Tub>miR-964) was
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significantly lower than control flies (Gal80ts; Tub-gal4) at 24 h post-infection (Fig. 1C).
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Whereas, the Drs expression in miR-964 knockout flies (miR-964 KO) was significantly
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higher than wide-type flies (w1118) both at 12 h and 24 h post-infection (Fig. 1D).
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Taken together, our results strongly argued that miR-964 negatively regulates Toll
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signaling immune response in Drosophila.
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3.2
In silico prediction of miR-964 targets
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To understand the molecular mechanism underlying the action of miR-964 in
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the Toll-mediated innate immune pathway, we carried an in silico genome-wide
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methods (TargetScan, miRanda, and PITA). By this means, we overlapped a total of
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367 targets of miR-964 by all three programs, which could significantly reduce the
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number of false positives of miRNA targets (Fig. 2A). Subsequently, we analyzed the
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367 miRNA-target relationships to screen potential target genes of miR-964 involved
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in Drosophila Toll signaling pathway. We finally identified two Toll signaling
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pathway-associated genes (Spz and Drs) (Fig. 2B). Both Spz and Drs are canonic
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components of Toll signaling pathway (Lemaitre et al., 1996). Spz is the extracellular
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cytokine which shares structural similarities with the nerve growth factor (NGF), and
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Drs has a marked susceptibility to fungal and Gram-positive bacterial infection. These
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above findings implied that miR-964 might influence two key genes (Spz and Drs) in
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Toll signaling pathway to regulate Drosophila innate immune response.
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Identification of miR-964 potential targets To investigate whether miR-964 could suppress Spz and Drs expression, we
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performed luciferase reporter assays in Drosophila S2 cells. The full-length 3’-UTR of
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Drosophila Spz and Drs mRNA were cloned and inserted into downstream of the
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firefly luciferase cDNA reporter vector, respectively (Fig. 3A). Our results
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demonstrated that miR-964 was unable to suppress the luciferase activity of the
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reporter with the 3’-UTR of Spz compared with the activity of the control reporter
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(Fig. 3B). However, miR-964 expression significantly reduced the activity of the
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luciferase reporter having the 3’-UTR of Drs by 50% compared to the pAc-empty
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control (Fig. 3C). This result seems to indicate that the Drs might be a target of
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miR-964. Besides, to further verify the target sites of the Drs 3’-UTR, we generated Drs
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3’-UTR mutant, in which the binding sites for seed sequences of miR-964 were
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mutated (Fig. 3A). The mutant reporter (Drs mut 3’-UTR) was not repressed by
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miR-964 expression in the co-transfection experiment (Fig. 3D). Overall, these results
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demonstrated that the 3’-UTR of Drs contains an operational binding site for miR-964
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to down-regulate. Therefore, we suggested that miR-964 might directly regulate the
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expression of Drs to involve in Toll signaling response in Drosophila.
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3.4
Drs is a direct target of miR-964
To further determine the effect of miR-964 targeting Drs on regulation of Toll
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innate immunity signaling in vivo, we experimental studied the mRNA levels of Spz
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and Dif after overexpression of miR-964. The results showed that miR-964
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overexpressing flies did not alter mRNA levels of Spz and Dif compared with controls
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both at 12 h and 24 h after M. luteus infection (Fig. 4A-B). Next, we further measured
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the expression of other AMPs induced by the Toll signaling pathway, including
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Defensin and Metchnikowin (Fig. S1). The results demonstrated that Defensin and
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Metchnikowin appeared to have no obvious changes compared to the control flies
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under the same circumstances. Therefore, we speculated that miR-964 only leads to
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the decrease of AMP Drs rather than other AMPs. Furthermore, western blotting
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analysis also showed that the protein expression level of Drs was significantly
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ACCEPTED MANUSCRIPT inhibited in miR-964 overexpressing flies at 24 h post-infection (Fig. 4C). Consistently,
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miR-964 knockout flies exhibited enhanced Toll signaling activity, and Drs expression
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was upregulated in miR-964 knockout flies at 24 h post-infection (Fig. 4D). Taken
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together, we concluded that miR-964 could negatively regulate Toll signaling
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response via controlling the expression of Drs.
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miR-964 sponge restores the immune phenotype of miR-964 overexpressing
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To further verify that miR-964 inhibits Toll signaling via targeting Drs, we
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simultaneously overexpressed miR-964 and a miR-964 sponge in adult flies. The
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miR-964 sponge could reduce miR-964 expression levels by soaking up miR-964
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molecules.
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Tub>miR-964+miR-964 sponge) rescues Drs mRNA expression, restoring it back to the
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level of controls (Fig. 5A). In addition, to monitor the differential effects of miR-964
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on the Drs expression in live flies, we also established Drs-green fluorescent protein
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(GFP) reporter transgenic flies. In agreement with the data from qRT-PCR assays,
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miR-964 overexpressing flies displayed a significantly lower GFP expression than
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control flies at 24 h post-infection (Fig. 5B). Similarly, miR-964 and miR-964 sponge
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co-expression also restored GFP expression level. These above results further
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confirmed that miR-964 inhibits Toll signal via directly targeting Drs.
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this
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4. Discussion
ACCEPTED MANUSCRIPT The immune system plays critical roles in maintaining homeostasis and
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defending against invading pathogens. To maintain homeostasis, the immune system
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must provide stability and withstand challenges. Targeted deletion of some miRNA
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genes can affect the homeostasis of the immune system. For example, loss of
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miR-155, miR-146a, and miR-223 all result in mild inflammatory responses in aged
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mice (Boldin et al., 2011; Johnnidis et al., 2008; Rodriguez et al., 2007; Thai et al.,
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2007). These results suggest that miRNA genes may be helpful for maintaining the
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homeostasis of the immune system. Recently our group has employed a transgenic
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UAS-miRNA library to identify miRNAs regulating Toll innate immunity signaling at a
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genome-scale (Li et al., 2017). This analysis has revealed that the direct effect of
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miRNA on the downstream AMP genes to regulate Toll innate immunity signaling is
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not unusual. Thus, we conclude that this regulation pattern could efficiently maintain
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the homeostasis of the immune system.
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In this study, we showed that miR-964 deficiency in flies leads to
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hyper-activation of AMP gene Drs and enhances survival rate of flies in the presence
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of immune challenge, and that over-expression of miR-964 compromises innate
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immunity. Furthermore, our analyses revealed that Drs is the direct target gene of
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miR-964. Besides Drs, our study also identifies other miR-964 target genes relevant to
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Toll signaling. However, we find that miR-964 does not repress the expression of
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other components in Toll signaling. Thus, our study uncovers miR-964 as a new
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component of a fine-tuning regulatory circuit that maintains the homeostasis of Toll
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signaling via directly inhibiting AMP gene.
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ACCEPTED MANUSCRIPT The miR-964 is encoded by the intron of CG31646 and belongs to the
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miR-959~964 cluster. A previous study also showed that the miR-959~964 cluster
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could inhibit immune function against pathogen and/or change the peak survival
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time (Vodala et al., 2012). While it is clear that miR-964 becomes one regulator in Toll
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innate immunity signaling, it appears that the underlying molecular mechanism of
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miR-959~964 cluster is rather complex. Moreover, the miR-959~964 cluster showed a
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similar phase and robust amplitude. Thus, they are probably encoded in a single
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transcription unit. It’s worth noting that the miR-959~964 cluster members have
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different seed sequences for each member. And that the miR-959~964 cluster has
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stronger effect on Toll innate immunity signaling than single miR-964. Therefore, it
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would be interesting to assess potential immune function of each member of the
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cluster against pathogens. However, how many immune genes are miR-959~964
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cluster’s targets and the extent of each miRNA’s contribution to Drosophila immune
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homeostasis remains an open question, which merits further investigation.
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In summary, our study enriches the repertoire of immune-modulating miRNAs,
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and provides new insights into the regulatory mechanism of Toll innate immunity
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signaling by miRNAs in Drosophila.
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Conflict of interest
All authors declare no conflicts of interest.
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Acknowledgements
ACCEPTED MANUSCRIPT This work was supported by the National Natural Science Foundation of China
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(No.31572324), the National Natural Science Youth Foundation of China (No.
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31501863), the Natural Science Research Project of Jiangsu Higher Education
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Institutions (No.16KJB180014) and the Priority Academic Program Development of
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Jiangsu Higher Education Institutions.
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References
296 297 298 299
M AN U
295
Aggarwal, K., Silverman, N., 2008. Positive and negative regulation of the Drosophila immune response. BMB Rep. 41, 267–277.
Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233.
Boldin, M.P., Taganov, K.D., Rao, D.S., Yang, L., Zhao, J.L., Kalwani, M., Garcia-Flores,
301
Y., Luong, M., Devrekanli, A., Xu, J., 2011. miR-146a is a significant brake on
302
autoimmunity, myeloproliferation, and cancer in mice. J. Exp. Med. 208,
303
1189–1201.
EP
305
Brennecke, J., Stark, A., Russell, R.B., Cohen, S.M., 2005. Principles of microRNA–target recognition. PLoS Biol 3, e85.
AC C
304
TE D
300
306
Chen, C., Schaffert, S., Fragoso, R., Loh, C., 2013. Regulation of immune responses
307
and tolerance: the microRNA perspective. Immunol. Rev. 253, 112–128.
308
Choi, I.K., Hyun, S., 2012. Conserved microRNA miR-8 in fat body regulates innate
309
immune homeostasis in Drosophila. Dev. Comp. Immunol. 37, 50–54.
310
De Gregorio, E., Spellman, P.T., Rubin, G.M., Lemaitre, B., 2001. Genome-wide
311
analysis of the Drosophila immune response by using oligonucleotide
312
microarrays. Proc. Natl. Acad. Sci. 98, 12590–12595.
313
De Gregorio, E., Spellman, P.T., Tzou, P., Rubin, G.M., Lemaitre, B., 2002. The Toll and
ACCEPTED MANUSCRIPT 314
Imd pathways are the major regulators of the immune response in Drosophila.
315
EMBO J 21, 2568–2579.
319 320 321 322 323 324
Garbuzov, A., Tatar, M., 2010. Hormonal regulation of Drosophila microRNA let-7
RI PT
318
targets in Drosophila. Genome Biol 5, R1.
and miR-125 that target innate immunity. Fly (Austin). 4, 306–311.
Huang, Y., Shen, X.J., Zou, Q., Wang, S.P., Tang, S.M., Zhang, G.Z., 2011. Biological functions of microRNAs: a review. J Physiol Biochem 67, 129–139.
Hultmark, D., 2003. Drosophila immunity: paths and patterns. Curr. Opin. Immunol.
SC
317
Enright, A.J., John, B., Gaul, U., Tuschl, T., Sander, C., Marks, D.S., 2003. MicroRNA
15, 12–19.
Ji, S., Sun, M., Zheng, X., Li, L., Sun, L., Chen, D., Sun, Q., 2014. Cell-surface
M AN U
316
325
localization of Pellino antagonizes Toll-mediated innate immune signalling by
326
controlling MyD88 turnover in Drosophila. Nat Commun 5. Johnnidis, J.B., Harris, M.H., Wheeler, R.T., Stehling-Sun, S., Lam, M.H., Kirak, O.,
328
Brummelkamp, T.R., Fleming, M.D., Camargo, F.D., 2008. Regulation of
329
progenitor cell proliferation and granulocyte function by microRNA-223. Nature
330
451, 1125–1129. doi:10.1038/nature06607
TE D
327
331
Kertesz, M., Iovino, N., Unnerstall, U., Gaul, U., Segal, E., 2007. The role of site
332
accessibility in microRNA target recognition. Nat. Genet. 39, 1278–1284.
335 336 337 338 339 340
EP
334
Lee, G.J., Hyun, S., 2014. Multiple targets of the microRNA miR-8 contribute to immune homeostasis in Drosophila. Dev. Comp. Immunol. 45, 245–251. Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster. Annu.
AC C
333
Rev. Immunol. 25, 697–743.
Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.-M., Hoffmann, J.A., 1996. The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983. Lemaitre, B., Reichhart, J.-M., Hoffmann, J.A., 1997. Drosophila host defense:
341
differential induction of antimicrobial peptide genes after infection by various
342
classes of microorganisms. Proc. Natl. Acad. Sci. 94, 14614–14619.
343
Lewis, B.P., Burge, C.B., Bartel, D.P., 2005. Conserved seed pairing, often flanked by
ACCEPTED MANUSCRIPT 344
adenosines, indicates that thousands of human genes are microRNA targets.
345
Cell 120, 15–20.
346 347
Li, L., Xu, J., Yang, D., Tan, X., Wang, H., 2010. Computational approaches for microRNA studies: a review. Mamm. Genome 21, 1–12. Li, S., Li, Y., Jin, P., Chen, L., Ma, F., 2016. miR-958 inhibits Toll signaling and
349
Drosomycin expression via directly targeting Toll and Dif in Drosophila
350
melanogaster. Am. J. Physiol. Physiol. ajpcell. 00251.2016.
351
RI PT
348
Li, Y., Li, S., Li, R., Xu, J., Jin, P., Chen, L., Ma, F., 2017. Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in
353
Drosophila melanogaster via co-targeting Drosomycin. Dev. Comp. Immunol. 68,
354
34–45.
356
M AN U
355
SC
352
Neyen, C., Bretscher, A.J., Binggeli, O., Lemaitre, B., 2014. Methods to study Drosophila immunity. Methods 68, 116–128.
Ragab, A., Buechling, T., Gesellchen, V., Spirohn, K., Boettcher, A., Boutros, M., 2011.
358
Drosophila Ras/MAPK signalling regulates innate immune responses in immune
359
and intestinal stem cells. EMBO J 30, 1123–1136.
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Rodriguez, A., Vigorito, E., Clare, S., Warren, M. V, Couttet, P., Soond, D.R., van
361
Dongen, S., Grocock, R.J., Das, P.P., Miska, E.A., Vetrie, D., Okkenhaug, K.,
362
Enright, A.J., Dougan, G., Turner, M., Bradley, A., 2007. Requirement of
363
bic/microRNA-155 for normal immune function. Science (80-. ). 316, 608–611.
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doi:10.1126/science.1139253
EP
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Ruby, J.G., Stark, A., Johnston, W.K., Kellis, M., Bartel, D.P., Lai, E.C., 2007. Evolution,
366
biogenesis, expression, and target predictions of a substantially expanded set of
367 368 369 370
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365
Drosophila microRNAs. Genome Res 17, 1850–1864.
Tanji, T., Ip, Y.T., 2005. Regulators of the Toll and Imd pathways in the Drosophila innate immune response. Trends Immunol 26, 193–198. Thai, T.H., Calado, D.P., Casola, S., Ansel, K.M., Xiao, C., Xue, Y., Murphy, A.,
371
Frendewey, D., Valenzuela, D., Kutok, J.L., Schmidt-Supprian, M., Rajewsky, N.,
372
Yancopoulos, G., Rao, A., Rajewsky, K., 2007. Regulation of the germinal center
373
response by microRNA-155. Science (80-. ). 316, 604–608.
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doi:10.1126/science.1141229 Valanne, S., Myllymäki, H., Kallio, J., Schmid, M.R., Kleino, A., Murumägi, A., Airaksinen, L., Kotipelto, T., Kaustio, M., Ulvila, J., 2010. Genome-wide RNA
377
interference in Drosophila cells identifies G protein-coupled receptor kinase 2
378
as a conserved regulator of NF-κB signaling. J. Immunol. 184, 6188–6198.
379
Valanne, S., Wang, J.-H., Rämet, M., 2011. The Drosophila toll signaling pathway. J.
380 381
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Immunol. 186, 649–656.
Vodala, S., Pescatore, S., Rodriguez, J., Buescher, M., Chen, Y.-W., Weng, R., Cohen, S.M., Rosbash, M., 2012. The oscillating miRNA 959-964 cluster impacts
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Drosophila feeding time and other circadian outputs. Cell Metab 16, 601–612.
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Xiong, X.-P., Kurthkoti, K., Chang, K.-Y., Li, J.-L., Ren, X., Ni, J.-Q., Rana, T.M., Zhou, R.,
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2016. miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.
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PLoS Pathog 12, e1006034.
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Fig. 1. miR-964 as a negative regulator of Toll signaling pathway
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(A) The flies were challenged with E. faecalis and the survival of the flies was
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monitored for 24 h. Wild-type flies (w1118) were used as a control. Wild-type flies
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(n=80); miR-964 overexpressing flies (n=69); miR-964 knockout flies (n=96). (B)
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Kinetics of expression of miR-964 in non-infected and M. luteus-infected flies.
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miR-964 overexpressing (C) or knockout (D) flies and controls were infected with M.
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luteus for 0 h, 12 h and 24 h and lysed for qRT-PCR analysis to measure mRNA levels
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of Drs. *P<0.05, **P<0.01.
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Fig. 2. Prediction of miR-964 targeted genes associated with Toll signaling in
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Drosophila.
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TargetScan, miRanda, and PITA software. A Venn diagram was created using the
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online software Draw Venn Diagram. (B) Software Cytoscape demonstrates miR-964
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targeted genes (Spz and Drs) associated with Toll signaling pathway.
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Fig. 3. miR-964 targets Drs, not Spz, in S2 cells.
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(A) The construct used to generate the 3’UTR reporter vectors for Spz and Drs.
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Mutant vector was constructed by replacing seed sequence components with the
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mutant gcggagc sequence. The interactions between miR-964 and its predicted
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target sequences in the 3’-UTRs of Spz (B), Drs (C), and Drs mut (D) were determined
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in Drosophila S2 cells. Forty-eight hours after transfection, cells were lysed for
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luciferase assays; firefly luciferase was normalized to Renilla luciferase activity. ns, no
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significant; ***P<0.001.
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Fig. 4. miR-964 inhibits expression of Drs in vivo.
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The mRNA expression levels of Spzl (A) and Dif (B) were determined in miR-964
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overexpressing flies and controls at 0, 12, and 24 h after M. luteus infection. Drs
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protein of miR-964 overexpressing (C) or knockout (D) flies and controls was
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measured at 0, 12, and 24 h post-infection.
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Fig. 5. miR-964 sponge restores Toll signaling in miR-964 overexpressing flies.
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(A) The levels of Drs were examined in control flies (Gal80ts; Tub-gal4),
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sponge co-expression flies (Gal80ts; Tub>miR-964+miR-964 sponge) at 24 h
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post-infection. (B) The fluorescence observed in miR-964 overexpressing fly (middle)
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shows less than the control fly (left) were infected with M. luteus for 24 h. Moreover,
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introduction of miR-964 sponge (right) restores GFP expression levels. ns, no
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significant; ***P<0.001.
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ACCEPTED MANUSCRIPT Highlights •
miR-964 fine-tunes over-activation of immune responses via negatively regulating Toll signaling in Drosophila.
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Overexpression or knockout of miR-964 alters the expression of antimicrobial
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Drosomycin is identified as the target of miR-964.
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S0145-305X(17)30355-5
DOI:
10.1016/j.dci.2017.08.008
Reference:
DCI 2963
To appear in:
Developmental and Comparative Immunology
Received Date: 6 July 2017 Revised Date:
15 August 2017
Accepted Date: 15 August 2017
Please cite this article as: Li, S., Xu, J., Sun, L., Li, R., Jin, P., Ma, F., Drosophila miR-964 modulates Toll signaling pathway in response to bacterial infection, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.08.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Drosophila miR-964 modulates Toll signaling pathway in response
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to bacterial infection
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Shengjie Li1, Jiao Xu1, Lianjie Sun1, Ruimin Li1, Ping Jin1 and Fei Ma1,*
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1 Laboratory for Comparative Genomics and Bioinformatics & Jiangsu Key Laboratory
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for Biodiversity and Biotechnology, College of Life Science, Nanjing Normal University,
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Nanjing 210046, China
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* To whom correspondence should be addressed. Tel: +86 25 85891852; Fax: +86 25
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85891852; Email: [email protected].
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Abstract
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Recent studies suggest that microRNA (miRNA) plays important roles in the
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control of immune response and tolerance. We previously found that the expression
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level of antimicrobial peptide gene Drosomycin (Drs) is decreased in miR-964
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overexpressing flies. Here, we further verified that miR-964 deficiency leads to
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hyper-activation of Drs. In addition, we employed three widely-used bioinformatic
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algorithms to screen potential miR-964 targets. Finally, we identified that miR-964
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modulates Toll signaling pathway, at least in part, by repressing the expression of Drs.
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Taken together, our study identifies miR-964 as a modulator of Toll signaling and
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enriches the repertoire of immune-modulating miRNAs in Drosophila.
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Key words: Toll signaling, miR-964, Drosomycin, Drosophila melanogaster
Abbreviations
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miRNA: microRNA
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TLRs: Toll-like receptors
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AMPs: antimicrobial peptides
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uc: unchallenged
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Drs: Drosomycin
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Spz: spätzle
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3’UTR: 3’-untranslated region
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M. luteus: Micrococcus luteus
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E. faecalis: Enterococcus faecalis
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1. Introduction
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The innate immune response is the first line of defense against microbial
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infections in both insects and mammals (Tanji and Ip, 2005). The Toll signaling
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pathway plays an important role in the Drosophila immune response, which includes
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the phagocytosis of microbes, the encapsulation and killing of parasites (Hultmark,
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2003). The identification of the Drosophila melanogaster Toll signaling pathway
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cascade and the subsequent characterization of Toll-like receptors (TLRs) have
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reshaped our understanding of the immune system (Valanne et al., 2011). It is
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well-known about the molecular components of the Drosophila Toll signaling
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ACCEPTED MANUSCRIPT pathway and their functions in immune response and tolerance against foreign
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pathogens (De Gregorio et al., 2002, 2001; Lemaitre et al., 1997). To date, the focus
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of most studies has been on protein-coding genes (Ji et al., 2014; Ragab et al., 2011).
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However, the discovery of miRNA has revealed an unexpected layer of genetic
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programs that regulate the Drosophila immune response at posttranscriptional levels
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(Chen et al., 2013).
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MiRNA, a class of small non-coding RNAs (~22 nucleotides), mainly regulates
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expression of specific gene through imperfect base pairing with the 3’-untranslated
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region (3’UTR) of mRNA (Bartel, 2009). MiRNA has widespread effects on gene
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expression by influencing target mRNA stability and/or translational efficiency
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(Brennecke et al., 2005). Emerging evidences indicate that miRNAs play important
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roles in impacting the intensity and/or duration of Drosophila immune signaling
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(Choi and Hyun, 2012; Garbuzov and Tatar, 2010; Lee and Hyun, 2014; Xiong et al.,
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2016). Especially, our recent works have demonstrated that miR-310 family
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participates in the Toll-mediated immune response (Li et al., 2017), and miR-958
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directly targets the Toll and Dif genes to negatively regulate the Toll signaling
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pathway (Li et al., 2016). Therefore, systematic analysis of the innate immune
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response in the model organism Drosophila has provided important insights into the
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mechanisms of immune regulation by miRNAs.
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In the Drosophila Toll signaling pathway, Gram-positive bacterial or fungal
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infection leads to the systemic production of antimicrobial peptides (AMPs)
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(Aggarwal and Silverman, 2008). Especially, the antimicrobial peptide Drosomycin (Drs)
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and Hoffmann, 2007). Our previous study showed that the level of AMP gene Drs is
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decreased in miR-964 overexpressing flies, suggesting that miR-964 might negatively
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regulate Toll signaling response in Drosophila (Li et al., 2017). To further prove our
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above hypothesis, in this study, we detailedly investigated the mechanism of miR-964
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regulating AMP expression in Drosophila. Our results revealed that mir-964 directly
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targets the AMP gene Drs to negatively regulate the Toll signaling immune response
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in Drosophila.
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2. Materials and methods
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2.1
Drosophila culture and stocks
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Drosophila stocks were maintained under standard culture conditions. All stocks
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in this study were obtained from the Bloomington Stock Center. The ubiquitous
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temperature sensitive Gal80ts-Gal4 driver system was used to express miRNAs of
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interest specifically in adult flies. Crosses were performed, and the resulting progeny
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were initially raised at the permissive temperature (18°C) for the inhibitor Gal80 to
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repress Gal4 activity. Following adult eclosion, flies were transferred to 29°C to allow
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activation of Gal4 in the adult stage.
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Infection and survival experiments
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Drosophila adult males, aged 3–4 days, were used for septic injury experiments.
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Control and miRNA-overexpressing flies were infected by Micrococcus luteus (M.
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immune response, to induce the expression of Drs. Septic injury was performed by
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pricking the thorax of the flies with a pulled glass capillary carrying M. luteus using a
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Nanoject apparatus (Nanoliter 2010, WPI) (Neyen et al., 2014). Flies were then
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incubated at 29°C and collected at appropriate time point post-infection. For the
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survival experiment, flies were then infected with Enterococcus faecalis (E. faecalis),
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and survival was monitored for 24 h (Valanne et al., 2010).
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RNA extraction and qRT-PCR
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Total RNA was extracted from control and treated adult flies using Trizol
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(Invitrogen). cDNA was synthesized using PrimeScript RT Master Mix kit (Takara).
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Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara) in triplicate on
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an ABI StepOne plus real-time PCR instrument. SYBR Prime Script miRNA RT-PCR kit
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(Takara) was used for quantitative RT-PCR of miRNAs. rp49 and 5s rRNA genes were
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used as reference controls. All qRT-PCR primer sequences are listed in Supplemental
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Table 1.
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In silico prediction of miRNA targets
Toll signaling pathway related genes that are potential targets of Drosophila
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miRNAs were identified using three different miRNA prediction algorithms, i.e.
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TargetScan, miRanda, and PITA (Huang et al., 2011; Li et al., 2010). TargetScan
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predicts biological targets of miRNAs by searching for the presence of conserved
ACCEPTED MANUSCRIPT 8mer and 7mer sites that match the seed region of each miRNA (Lewis et al., 2005;
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Ruby et al., 2007). miRanda determines interactions through free-energy dynamics
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across the entire miRNA : mRNA interaction, with increased weight given to the
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miRNA 5’ seed region (Enright et al., 2003). PITA requires sequence similarity within
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the seed region but also determines the accessibility of the mRNA 3’-UTR based on
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free energy (Kertesz et al., 2007).
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2.5
Plasmid construction and luciferase assay
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A miR-964 expression plasmid was constructed from the empty pAc5.1 vector
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(Promega). The 3’-UTR reporter plasmids for spätzle (Spz) and Drs were generated by
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inserting PCR-amplified 3’-UTR fragments downstream of the luciferase gene in the
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pAc5.1 luciferase vector. Binding-site mutant construct was generated via Fast
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Site-Directed Mutagenesis Kit (TIANGEN). Transient transfections were performed in
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S2 cells using X-treme GENE HP transfection reagent (Roche). For dual-luciferase
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reporter assays, cells were harvested at 48 h post-transfection. Firefly and Renilla
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luciferase activity were measured using the Dual-Luciferase Assay Reporter System
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(Promega). Renilla luciferase was used to normalize the transfection efficiency.
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Western blotting
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For western blotting, the anti-β-actin primary antibody (Bioworld) was used at a
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1:2,000 dilution with an anti-rabbit HRP-conjugated secondary antibody (Vazyme) at
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1:30,000. The antibody against Drs was generated by immunizing rabbits with the
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Escherichia coli-produced recombinant His6-Drs (LSGRYKGPCAVWDNET).
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2.7
Statistical analysis All experimental data in this study were collected from three biological
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replicates. All statistical analyses were shown as means ± SD. Significant differences
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between two groups were determined by two-tailed Student’s t-test. Statistical
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analysis of fly survival experiments was carried out using the log-rank (Mantel-Cox)
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test. For all tests, a P value < 0.05 was considered statistically significant. ns, not
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significant, *P < 0.05; **P < 0.01; ***P < 0.001.
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3. Results
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3.1 miR-964 is involved in negatively regulating the Drosophila Toll signaling
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pathway
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The expression level of Drs is decreased in miR-964 overexpressing flies in our
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previous work (Li et al., 2017), so we speculate that Toll signaling might be
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moderately regulated by miR-964. To further verify whether miR-964 modulates
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Drosophila immune response, we used septic injury with Gram-positive bacteria (E.
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faecalis) to examine the effect of miR-964 on the fly’s survival (Fig. 1A). The miR-964
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overexpressing flies (Gal80ts; Tub>miR-964), control flies (w1118) and miR-964
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knockout flies (miR-964 KO) were infected by pricking with E. faecalis, respectively.
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The miR-964 overexpressing flies showed a statistically significant reduction in
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survival compared with both the control flies and miR-964 knockout flies. However,
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worth noting that the miR-964 knockout flies had a more significant effect on fly’s
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survival than control flies by compared with the miR-964 overexpressing flies. These
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findings suggested that miR-964 might influence the host defense ability against
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Gram-positive bacterial infection. Next, temporal expression patterns of miR-964
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were analyzed following exposure to other bacterial (M. luteus) compared with the
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physical injury control (Fig. 1B). Our results revealed that the expression of miR-964
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in flies infected by M. luteus was significantly higher than the injured flies at 24 h, 48
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h and 72 h after challenge, indicating that Drosophila host defense causes the
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fluctuation of expression level of endogenous miR-964. We further infected miR-964
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overexpressing flies and miR-964 knockout flies with M. luteus, and tested the
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expression level of Drs at 0 h, 12 h, 24 h post-infection by qRT-PCR. As expected, the
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Drs expression in miR-964 overexpressing flies (Gal80ts; Tub>miR-964) was
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significantly lower than control flies (Gal80ts; Tub-gal4) at 24 h post-infection (Fig. 1C).
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Whereas, the Drs expression in miR-964 knockout flies (miR-964 KO) was significantly
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higher than wide-type flies (w1118) both at 12 h and 24 h post-infection (Fig. 1D).
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Taken together, our results strongly argued that miR-964 negatively regulates Toll
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signaling immune response in Drosophila.
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In silico prediction of miR-964 targets
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To understand the molecular mechanism underlying the action of miR-964 in
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the Toll-mediated innate immune pathway, we carried an in silico genome-wide
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methods (TargetScan, miRanda, and PITA). By this means, we overlapped a total of
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367 targets of miR-964 by all three programs, which could significantly reduce the
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number of false positives of miRNA targets (Fig. 2A). Subsequently, we analyzed the
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367 miRNA-target relationships to screen potential target genes of miR-964 involved
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in Drosophila Toll signaling pathway. We finally identified two Toll signaling
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pathway-associated genes (Spz and Drs) (Fig. 2B). Both Spz and Drs are canonic
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components of Toll signaling pathway (Lemaitre et al., 1996). Spz is the extracellular
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cytokine which shares structural similarities with the nerve growth factor (NGF), and
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Drs has a marked susceptibility to fungal and Gram-positive bacterial infection. These
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above findings implied that miR-964 might influence two key genes (Spz and Drs) in
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Toll signaling pathway to regulate Drosophila innate immune response.
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Identification of miR-964 potential targets To investigate whether miR-964 could suppress Spz and Drs expression, we
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performed luciferase reporter assays in Drosophila S2 cells. The full-length 3’-UTR of
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Drosophila Spz and Drs mRNA were cloned and inserted into downstream of the
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firefly luciferase cDNA reporter vector, respectively (Fig. 3A). Our results
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demonstrated that miR-964 was unable to suppress the luciferase activity of the
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reporter with the 3’-UTR of Spz compared with the activity of the control reporter
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(Fig. 3B). However, miR-964 expression significantly reduced the activity of the
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luciferase reporter having the 3’-UTR of Drs by 50% compared to the pAc-empty
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control (Fig. 3C). This result seems to indicate that the Drs might be a target of
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miR-964. Besides, to further verify the target sites of the Drs 3’-UTR, we generated Drs
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3’-UTR mutant, in which the binding sites for seed sequences of miR-964 were
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mutated (Fig. 3A). The mutant reporter (Drs mut 3’-UTR) was not repressed by
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miR-964 expression in the co-transfection experiment (Fig. 3D). Overall, these results
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demonstrated that the 3’-UTR of Drs contains an operational binding site for miR-964
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to down-regulate. Therefore, we suggested that miR-964 might directly regulate the
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expression of Drs to involve in Toll signaling response in Drosophila.
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Drs is a direct target of miR-964
To further determine the effect of miR-964 targeting Drs on regulation of Toll
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innate immunity signaling in vivo, we experimental studied the mRNA levels of Spz
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and Dif after overexpression of miR-964. The results showed that miR-964
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overexpressing flies did not alter mRNA levels of Spz and Dif compared with controls
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both at 12 h and 24 h after M. luteus infection (Fig. 4A-B). Next, we further measured
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the expression of other AMPs induced by the Toll signaling pathway, including
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Defensin and Metchnikowin (Fig. S1). The results demonstrated that Defensin and
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Metchnikowin appeared to have no obvious changes compared to the control flies
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under the same circumstances. Therefore, we speculated that miR-964 only leads to
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the decrease of AMP Drs rather than other AMPs. Furthermore, western blotting
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analysis also showed that the protein expression level of Drs was significantly
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miR-964 knockout flies exhibited enhanced Toll signaling activity, and Drs expression
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was upregulated in miR-964 knockout flies at 24 h post-infection (Fig. 4D). Taken
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together, we concluded that miR-964 could negatively regulate Toll signaling
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response via controlling the expression of Drs.
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miR-964 sponge restores the immune phenotype of miR-964 overexpressing
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To further verify that miR-964 inhibits Toll signaling via targeting Drs, we
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simultaneously overexpressed miR-964 and a miR-964 sponge in adult flies. The
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miR-964 sponge could reduce miR-964 expression levels by soaking up miR-964
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molecules.
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Tub>miR-964+miR-964 sponge) rescues Drs mRNA expression, restoring it back to the
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level of controls (Fig. 5A). In addition, to monitor the differential effects of miR-964
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on the Drs expression in live flies, we also established Drs-green fluorescent protein
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(GFP) reporter transgenic flies. In agreement with the data from qRT-PCR assays,
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miR-964 overexpressing flies displayed a significantly lower GFP expression than
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control flies at 24 h post-infection (Fig. 5B). Similarly, miR-964 and miR-964 sponge
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co-expression also restored GFP expression level. These above results further
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confirmed that miR-964 inhibits Toll signal via directly targeting Drs.
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defending against invading pathogens. To maintain homeostasis, the immune system
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must provide stability and withstand challenges. Targeted deletion of some miRNA
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genes can affect the homeostasis of the immune system. For example, loss of
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miR-155, miR-146a, and miR-223 all result in mild inflammatory responses in aged
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mice (Boldin et al., 2011; Johnnidis et al., 2008; Rodriguez et al., 2007; Thai et al.,
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2007). These results suggest that miRNA genes may be helpful for maintaining the
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homeostasis of the immune system. Recently our group has employed a transgenic
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UAS-miRNA library to identify miRNAs regulating Toll innate immunity signaling at a
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genome-scale (Li et al., 2017). This analysis has revealed that the direct effect of
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miRNA on the downstream AMP genes to regulate Toll innate immunity signaling is
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not unusual. Thus, we conclude that this regulation pattern could efficiently maintain
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the homeostasis of the immune system.
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In this study, we showed that miR-964 deficiency in flies leads to
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hyper-activation of AMP gene Drs and enhances survival rate of flies in the presence
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of immune challenge, and that over-expression of miR-964 compromises innate
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immunity. Furthermore, our analyses revealed that Drs is the direct target gene of
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miR-964. Besides Drs, our study also identifies other miR-964 target genes relevant to
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Toll signaling. However, we find that miR-964 does not repress the expression of
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other components in Toll signaling. Thus, our study uncovers miR-964 as a new
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component of a fine-tuning regulatory circuit that maintains the homeostasis of Toll
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signaling via directly inhibiting AMP gene.
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ACCEPTED MANUSCRIPT The miR-964 is encoded by the intron of CG31646 and belongs to the
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miR-959~964 cluster. A previous study also showed that the miR-959~964 cluster
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could inhibit immune function against pathogen and/or change the peak survival
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time (Vodala et al., 2012). While it is clear that miR-964 becomes one regulator in Toll
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innate immunity signaling, it appears that the underlying molecular mechanism of
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miR-959~964 cluster is rather complex. Moreover, the miR-959~964 cluster showed a
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similar phase and robust amplitude. Thus, they are probably encoded in a single
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transcription unit. It’s worth noting that the miR-959~964 cluster members have
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different seed sequences for each member. And that the miR-959~964 cluster has
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stronger effect on Toll innate immunity signaling than single miR-964. Therefore, it
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would be interesting to assess potential immune function of each member of the
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cluster against pathogens. However, how many immune genes are miR-959~964
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cluster’s targets and the extent of each miRNA’s contribution to Drosophila immune
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homeostasis remains an open question, which merits further investigation.
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In summary, our study enriches the repertoire of immune-modulating miRNAs,
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and provides new insights into the regulatory mechanism of Toll innate immunity
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signaling by miRNAs in Drosophila.
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Conflict of interest
All authors declare no conflicts of interest.
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Acknowledgements
ACCEPTED MANUSCRIPT This work was supported by the National Natural Science Foundation of China
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(No.31572324), the National Natural Science Youth Foundation of China (No.
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31501863), the Natural Science Research Project of Jiangsu Higher Education
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Institutions (No.16KJB180014) and the Priority Academic Program Development of
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Jiangsu Higher Education Institutions.
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References
296 297 298 299
M AN U
295
Aggarwal, K., Silverman, N., 2008. Positive and negative regulation of the Drosophila immune response. BMB Rep. 41, 267–277.
Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233.
Boldin, M.P., Taganov, K.D., Rao, D.S., Yang, L., Zhao, J.L., Kalwani, M., Garcia-Flores,
301
Y., Luong, M., Devrekanli, A., Xu, J., 2011. miR-146a is a significant brake on
302
autoimmunity, myeloproliferation, and cancer in mice. J. Exp. Med. 208,
303
1189–1201.
EP
305
Brennecke, J., Stark, A., Russell, R.B., Cohen, S.M., 2005. Principles of microRNA–target recognition. PLoS Biol 3, e85.
AC C
304
TE D
300
306
Chen, C., Schaffert, S., Fragoso, R., Loh, C., 2013. Regulation of immune responses
307
and tolerance: the microRNA perspective. Immunol. Rev. 253, 112–128.
308
Choi, I.K., Hyun, S., 2012. Conserved microRNA miR-8 in fat body regulates innate
309
immune homeostasis in Drosophila. Dev. Comp. Immunol. 37, 50–54.
310
De Gregorio, E., Spellman, P.T., Rubin, G.M., Lemaitre, B., 2001. Genome-wide
311
analysis of the Drosophila immune response by using oligonucleotide
312
microarrays. Proc. Natl. Acad. Sci. 98, 12590–12595.
313
De Gregorio, E., Spellman, P.T., Tzou, P., Rubin, G.M., Lemaitre, B., 2002. The Toll and
ACCEPTED MANUSCRIPT 314
Imd pathways are the major regulators of the immune response in Drosophila.
315
EMBO J 21, 2568–2579.
319 320 321 322 323 324
Garbuzov, A., Tatar, M., 2010. Hormonal regulation of Drosophila microRNA let-7
RI PT
318
targets in Drosophila. Genome Biol 5, R1.
and miR-125 that target innate immunity. Fly (Austin). 4, 306–311.
Huang, Y., Shen, X.J., Zou, Q., Wang, S.P., Tang, S.M., Zhang, G.Z., 2011. Biological functions of microRNAs: a review. J Physiol Biochem 67, 129–139.
Hultmark, D., 2003. Drosophila immunity: paths and patterns. Curr. Opin. Immunol.
SC
317
Enright, A.J., John, B., Gaul, U., Tuschl, T., Sander, C., Marks, D.S., 2003. MicroRNA
15, 12–19.
Ji, S., Sun, M., Zheng, X., Li, L., Sun, L., Chen, D., Sun, Q., 2014. Cell-surface
M AN U
316
325
localization of Pellino antagonizes Toll-mediated innate immune signalling by
326
controlling MyD88 turnover in Drosophila. Nat Commun 5. Johnnidis, J.B., Harris, M.H., Wheeler, R.T., Stehling-Sun, S., Lam, M.H., Kirak, O.,
328
Brummelkamp, T.R., Fleming, M.D., Camargo, F.D., 2008. Regulation of
329
progenitor cell proliferation and granulocyte function by microRNA-223. Nature
330
451, 1125–1129. doi:10.1038/nature06607
TE D
327
331
Kertesz, M., Iovino, N., Unnerstall, U., Gaul, U., Segal, E., 2007. The role of site
332
accessibility in microRNA target recognition. Nat. Genet. 39, 1278–1284.
335 336 337 338 339 340
EP
334
Lee, G.J., Hyun, S., 2014. Multiple targets of the microRNA miR-8 contribute to immune homeostasis in Drosophila. Dev. Comp. Immunol. 45, 245–251. Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster. Annu.
AC C
333
Rev. Immunol. 25, 697–743.
Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.-M., Hoffmann, J.A., 1996. The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983. Lemaitre, B., Reichhart, J.-M., Hoffmann, J.A., 1997. Drosophila host defense:
341
differential induction of antimicrobial peptide genes after infection by various
342
classes of microorganisms. Proc. Natl. Acad. Sci. 94, 14614–14619.
343
Lewis, B.P., Burge, C.B., Bartel, D.P., 2005. Conserved seed pairing, often flanked by
ACCEPTED MANUSCRIPT 344
adenosines, indicates that thousands of human genes are microRNA targets.
345
Cell 120, 15–20.
346 347
Li, L., Xu, J., Yang, D., Tan, X., Wang, H., 2010. Computational approaches for microRNA studies: a review. Mamm. Genome 21, 1–12. Li, S., Li, Y., Jin, P., Chen, L., Ma, F., 2016. miR-958 inhibits Toll signaling and
349
Drosomycin expression via directly targeting Toll and Dif in Drosophila
350
melanogaster. Am. J. Physiol. Physiol. ajpcell. 00251.2016.
351
RI PT
348
Li, Y., Li, S., Li, R., Xu, J., Jin, P., Chen, L., Ma, F., 2017. Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in
353
Drosophila melanogaster via co-targeting Drosomycin. Dev. Comp. Immunol. 68,
354
34–45.
356
M AN U
355
SC
352
Neyen, C., Bretscher, A.J., Binggeli, O., Lemaitre, B., 2014. Methods to study Drosophila immunity. Methods 68, 116–128.
Ragab, A., Buechling, T., Gesellchen, V., Spirohn, K., Boettcher, A., Boutros, M., 2011.
358
Drosophila Ras/MAPK signalling regulates innate immune responses in immune
359
and intestinal stem cells. EMBO J 30, 1123–1136.
TE D
357
Rodriguez, A., Vigorito, E., Clare, S., Warren, M. V, Couttet, P., Soond, D.R., van
361
Dongen, S., Grocock, R.J., Das, P.P., Miska, E.A., Vetrie, D., Okkenhaug, K.,
362
Enright, A.J., Dougan, G., Turner, M., Bradley, A., 2007. Requirement of
363
bic/microRNA-155 for normal immune function. Science (80-. ). 316, 608–611.
364
doi:10.1126/science.1139253
EP
360
Ruby, J.G., Stark, A., Johnston, W.K., Kellis, M., Bartel, D.P., Lai, E.C., 2007. Evolution,
366
biogenesis, expression, and target predictions of a substantially expanded set of
367 368 369 370
AC C
365
Drosophila microRNAs. Genome Res 17, 1850–1864.
Tanji, T., Ip, Y.T., 2005. Regulators of the Toll and Imd pathways in the Drosophila innate immune response. Trends Immunol 26, 193–198. Thai, T.H., Calado, D.P., Casola, S., Ansel, K.M., Xiao, C., Xue, Y., Murphy, A.,
371
Frendewey, D., Valenzuela, D., Kutok, J.L., Schmidt-Supprian, M., Rajewsky, N.,
372
Yancopoulos, G., Rao, A., Rajewsky, K., 2007. Regulation of the germinal center
373
response by microRNA-155. Science (80-. ). 316, 604–608.
ACCEPTED MANUSCRIPT 374 375
doi:10.1126/science.1141229 Valanne, S., Myllymäki, H., Kallio, J., Schmid, M.R., Kleino, A., Murumägi, A., Airaksinen, L., Kotipelto, T., Kaustio, M., Ulvila, J., 2010. Genome-wide RNA
377
interference in Drosophila cells identifies G protein-coupled receptor kinase 2
378
as a conserved regulator of NF-κB signaling. J. Immunol. 184, 6188–6198.
379
Valanne, S., Wang, J.-H., Rämet, M., 2011. The Drosophila toll signaling pathway. J.
380 381
RI PT
376
Immunol. 186, 649–656.
Vodala, S., Pescatore, S., Rodriguez, J., Buescher, M., Chen, Y.-W., Weng, R., Cohen, S.M., Rosbash, M., 2012. The oscillating miRNA 959-964 cluster impacts
383
Drosophila feeding time and other circadian outputs. Cell Metab 16, 601–612.
SC
382
Xiong, X.-P., Kurthkoti, K., Chang, K.-Y., Li, J.-L., Ren, X., Ni, J.-Q., Rana, T.M., Zhou, R.,
385
2016. miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.
386
PLoS Pathog 12, e1006034.
387 388
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Fig. 1. miR-964 as a negative regulator of Toll signaling pathway
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(A) The flies were challenged with E. faecalis and the survival of the flies was
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monitored for 24 h. Wild-type flies (w1118) were used as a control. Wild-type flies
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(n=80); miR-964 overexpressing flies (n=69); miR-964 knockout flies (n=96). (B)
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Kinetics of expression of miR-964 in non-infected and M. luteus-infected flies.
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miR-964 overexpressing (C) or knockout (D) flies and controls were infected with M.
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luteus for 0 h, 12 h and 24 h and lysed for qRT-PCR analysis to measure mRNA levels
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of Drs. *P<0.05, **P<0.01.
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Fig. 2. Prediction of miR-964 targeted genes associated with Toll signaling in
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Drosophila.
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TargetScan, miRanda, and PITA software. A Venn diagram was created using the
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online software Draw Venn Diagram. (B) Software Cytoscape demonstrates miR-964
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targeted genes (Spz and Drs) associated with Toll signaling pathway.
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Fig. 3. miR-964 targets Drs, not Spz, in S2 cells.
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(A) The construct used to generate the 3’UTR reporter vectors for Spz and Drs.
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Mutant vector was constructed by replacing seed sequence components with the
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mutant gcggagc sequence. The interactions between miR-964 and its predicted
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target sequences in the 3’-UTRs of Spz (B), Drs (C), and Drs mut (D) were determined
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in Drosophila S2 cells. Forty-eight hours after transfection, cells were lysed for
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luciferase assays; firefly luciferase was normalized to Renilla luciferase activity. ns, no
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significant; ***P<0.001.
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Fig. 4. miR-964 inhibits expression of Drs in vivo.
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The mRNA expression levels of Spzl (A) and Dif (B) were determined in miR-964
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overexpressing flies and controls at 0, 12, and 24 h after M. luteus infection. Drs
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protein of miR-964 overexpressing (C) or knockout (D) flies and controls was
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measured at 0, 12, and 24 h post-infection.
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Fig. 5. miR-964 sponge restores Toll signaling in miR-964 overexpressing flies.
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(A) The levels of Drs were examined in control flies (Gal80ts; Tub-gal4),
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sponge co-expression flies (Gal80ts; Tub>miR-964+miR-964 sponge) at 24 h
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post-infection. (B) The fluorescence observed in miR-964 overexpressing fly (middle)
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shows less than the control fly (left) were infected with M. luteus for 24 h. Moreover,
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introduction of miR-964 sponge (right) restores GFP expression levels. ns, no
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significant; ***P<0.001.
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ACCEPTED MANUSCRIPT Highlights •
miR-964 fine-tunes over-activation of immune responses via negatively regulating Toll signaling in Drosophila.
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Overexpression or knockout of miR-964 alters the expression of antimicrobial
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Drosomycin is identified as the target of miR-964.
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peptide Drosomycin after Micrococcus luteus infection.