The Regulation Of MicroRNA In The Immune Response Of Drosophila Toll Signaling

Drosophila melanogaster is a well-established model system used to decipher the molecular and cellular mechanisms that underlie complex biological processes.Fruit flies neutralize potentially harmful microorganisms by producing antimicrobial peptides (AMPs). The expression of AMPs is mainly regulated by two signaling pathways: Toll and immune deficiency (Imd), which show striking similarities to the mammalian Toll-like receptor/interleukin-1 (TLR) and tumor necrosis factor-?pathways (TNF-?), respectively. It is clear that the Toll signaling pathway in Drosophila plays a key role in the innate immune response against Gram-positive bacterial and fungal infections.MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate protein synthesis by base-pairing to the 3'-untranslated regions (3'UTR) of target mRNAs. Over-activation of the immune response can negatively impact individual fitness, and therefore safeguard mechanisms must be put in place to either tolerate or limit the potential for overactive immune responses. Post-transcriptional gene regulation has been proposed as a mechanism to fine-tune immune responses and other physiological processes to prevent or limit over-activation. Recent studies have demonstrated that miRNAs extensively modulate immune responses against bacteria and parasites across different species. Although Toll signaling pathway is well-studied, the precise mechanisms of post-transcriptional regulation of key components of the Toll signaling pathway by miRNAs remain obscure. To better understand the role of miRNAs in regulation of Toll signaling pathway, following studies were done:1. Small RNA-seq was performed on total RNA from wild-type files at 3, 12 and 24 h post-infection with either PBS or M. luteus (a Gram-positive bacterium). In total,445 miRNAs were identified as candidate miRNAs regulating the immune response of Drosophila upon M. luteus infection. Only 6 and 14 miRNAs were differentially expressed in M. luteus infected flies compared to PBS treated flies at 3 h and 12 h post-infection, respectively. While 86 differentially expressed miRNAs were identified in M. luteus infected flies compared to PBS treated flies.2. Following Gram-positive bacterial challenge, we identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related. Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 immune response related miRNAs. These eight miRNAs are miR-310 family (miR-310,miR-311, miR-312, miR-313) , miR-317, miR-963, miR-964 and miR-958.3. The mechanism of regulation of miR-958 and miR-310 family on the Toll signaling pathway of Drosophila melanogaster has been studied in depth. Using three target gene prediction software (miRanda, PITA and Targetscan), we predicted the target genes of miR-958 and miR-310 family in the whole genome of Drosophila.Based on further analysis of the predicted results, we identified the potential target genes Toll and Dif of miR-958-regulated Toll signaling pathway, and the potential target genes Toll,Cactus and Drosomycin of miR-310 family-regulated Toll signaling pathway.4. The potential target genes of miR-958 and miR-310 family were identified by luciferase reporter assay. The results show that miR-958 could interact with the target genes Toll and Dif, while the miR-310 family could interact only with the target gene Drosomycin, but could not interact with the other two target genes Toll and Cactus.5. Detection by qRT-PCR and Western Blotting,the effect of miR-310?313 family on the regulation of Drosophila Toll signal pathway is stronger than single miR-310 family. While the expression of miR-958 has a significant inhibitory effect on the expression of Toll and Dif protein. The regulation of multiple target genes also reflects the abundant spatial and hierarchical properties of miRNA-regulated Toll signaling pathway.The results of this study show that miRNAs play an important role in the immune response of Toll signaling pathway in Drosophila melanogaster. We believe that the miR-310 family members directly target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. While miR-958 inhibits the key genes Toll and Dif of Toll signaling pathway and indirectly negatively regulates the expression of the antimicrobial peptide Drosomycin. Taken together,these results not only revealed a novel function and modulation pattern of miR-958 and miR-310 family members, but also provided a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.