Multiple targets of the microRNA miR-8 contribute to immune homeostasis in Drosophila
Introduction
MicroRNAs (miRNAs) are small noncoding 22-nucleotide(nt)-long RNAs that posttranscriptionally repress protein synthesis from their target mRNAs by base-pairing to the 3′-untranslated regions (UTR) (Bartel, 2009, Brennecke et al., 2005). They thus exert significantly important roles in various biological processes, such as cell growth/proliferation, development, immunity, and metabolism. As with coding genes, there are orthologs of many miRNAs among a broad range of species, and some conserved miRNAs have been reported to have similar physiological roles in different species (Bushati and Cohen, 2007, Ibanez-Ventoso et al., 2008).
miR-8 is a highly conserved miRNA broadly found in bilateria. In humans, there are five paralogs, known as the miR-200 family of miRNAs. It is well known that the miR-200 family modulates the process of epithelial to mesenchymal transition (EMT), and it is closely associated with cancer development (Creighton et al., 2013, Zhang et al., 2013). In many cancers, miR-200 has been shown to suppress the initial step of metastasis by inhibiting EMT, although in ovarian cancer, miR-200 promotes tumorigenesis by controlling oxidative stress responses (Mateescu et al., 2011). miR-8 in Drosophila is known to regulate Wnt and insulin signaling, which are also known to be regulated by miR-200 in mammals (Kennell et al., 2008). Recently, Drosophila miRNA has been predicted to be involved in the immune response pathway (Fullaondo and Lee, 2011), and miR-8 miRNA has been shown to play a role in maintaining basal levels of immune activity in Drosophila (Choi and Hyun, 2012).
Drosophila has an innate immune system to protect against invading microorganisms such as bacteria, fungi, and viruses (Ferrandon et al., 2007). By producing antimicrobial peptides (AMPs), flies neutralize potentially harmful microorganisms. Innate immune signaling pathways leading to AMP expression in Drosophila have been well elucidated. Pathogenic microorganisms invading the body of Drosophila encounter humoral or transmembrane proteins, called pattern recognition proteins, that sense specific components of bacterial cell walls. Recognition of pathogenic components by pattern recognition proteins triggers the two main signaling pathways of the innate immune response, the Toll and Imd pathways (Ferrandon et al., 2007, Minakhina and Steward, 2006, Valanne et al., 2011, Wang and Ligoxygakis, 2006). The Toll pathway is mainly activated by infections with fungi and gram-positive bacteria, whereas the Imd pathway is largely induced by gram-negative bacteria. However, recent studies indicate that activation of these pathways is not mutually exclusive, but in fact, interdependent (Gottar et al., 2006, Lemaitre et al., 1996, Lemaitre et al., 1997, Tanji et al., 2007). Recognition of infectious fungi or gram-positive bacteria induces processing of the humoral ligand, Spaetzle, which binds to and activates the transmembrane receptor, Toll. This triggers the Toll immune pathway cascade, finally leading to activation of the NF-κB family of transcription factors, Dif and Dorsal. Activation of Dif and Dorsal by the Toll immune pathway then induces the expression of a class of AMPs like Drosomycin (Ferrandon et al., 2007, Minakhina and Steward, 2006, Valanne et al., 2011, Wang and Ligoxygakis, 2006). Activation of the Imd immune pathway by gram-negative bacteria induces nuclear localization of the NF-κB family transcription factor Relish, leading to the expression of various AMPs such as Diptericin (Ferrandon et al., 2007, Minakhina and Steward, 2006, Valanne et al., 2011, Wang and Ligoxygakis, 2006). The fat body, the fly counterpart of the liver and adipose tissue, is the main producer of AMPs in response to infection by pathogens. AMPs emanating from the fat body circulate systemically in the fly hemolymph to prevent pathogenesis (Ferrandon et al., 2007, Minakhina and Steward, 2006, Valanne et al., 2011, Wang and Ligoxygakis, 2006).
In addition to the fat body, several Drosophila tissues are known to express AMPs (Ferrandon et al., 2007). Flies harboring a Drosomycin-GFP reporter express GFP signals in various epithelial tissues in the absence of septic injuries (Ferrandon et al., 1998). Moreover, a recent study showed that AMP expression can be induced via a pathway independent of Toll or Imd in response to stressful environmental conditions (Becker et al., 2010). Understanding the physiological significance of these examples of basal AMP expression requires further investigation.
We previously found that the levels of AMPs such as Drosomycin and Diptericin are elevated in miR-8-null flies, indicating that miR-8 suppresses basal immune activity (Choi and Hyun, 2012). Here, we further discovered that miR-8 targets multiple genes coding for immunity, contributing to miR-8-mediated homeostasis in the immune response. We found Toll and Dorsal, the main components of the Toll immune pathway, to be miR-8 targets, and their transcript levels to be elevated in the fat bodies of miR-8-null flies. We also found that elevated expression of U-shaped (Ush), a previously verified miR-8 target, can increase the level of Drosomycin. Finally, the perturbed immune homeostasis and lethal phenotypes of miR-8 null flies are rescued by reducing the activity of Toll pathway or Ush, indicating that miR-8 control of basal immune homeostasis is potentially beneficial for fly survival.
Section snippets
Fly strains
The miR-8 null mutant (mir-8△2) and UAS-mir-8, UAS-Ush, Ush1513 and ppl Gal4 mutant flies are described elsewhere (Hyun et al., 2009). Spzrm7 fly is described elsewhere (Tzou et al., 2002). UAS-Dorsal flies were obtained from the Bloomington Drosophila Stock Center, and UAS-Dorsal RNAi flies were from the Vienna Drosophila RNAi Center.
Fly rearing
Flies were maintained at 25 °C with 60% humidity and 12-h light and dark cycles on standard glucose medium: 862 g glucose, 408 g cornmeal, 624 g dried yeast, and 93 g
Toll and Dorsal genes are miR-8 targets
Because Drosomycin level is increased in miR-8 null flies (Choi and Hyun, 2012), we speculated that the Toll pathway might be moderately enhanced. Interestingly, two genes in the Toll pathway were predicted to be miR-8 targets by the miTarget program (Kim et al., 2006). The genes were Toll and Dorsal. miTarget predicted one canonical and one noncanonical target site for miR-8 on the Toll 3′-UTR, and two noncanonical target sites for miR-8 on the Dorsal 3′-UTR (Fig. 1a). To verify that the Toll
Discussion
miRNAs are small, non-coding RNAs that modulate the expression of their target genes by binding to partially complementary sites in the 3′-UTR of the target mRNA. As such, miRNAs play significantly important roles in almost all biological processes through posttranscriptional regulation of gene expression. Moreover, the expression of miRNA is heavily regulated by various biological conditions and, together with transcription factors, constitutes the nodes of a network of genome-wide gene
Acknowledgments
We are very grateful to Dr. Jin Wu Nam for the analysis of the target prediction of miR-8 microRNA. We thank the Bloomington Drosophila Stock Center and the Vienna Drosophila RNAi Center for fly stocks used in this study. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2012R1A1A1009732 to S.H.).
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