LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis

Abstract

Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. In this study, we firstly discover that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, our results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, our findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w¹¹¹⁸) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, our study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity.

Keywords: Drosophila melanogaster; NF-κB transcription factor Dif/Dorsal; antimicrobial peptides; innate immunity; lncRNA-CR11538; toll pathway.

Figure 1

LncRNA-CR11538 is highly expressed after M. luteus stimulation. (A) Volcano map showing these differentially expressed lncRNAs in RNA-seq between PBS-treated and M. luteus-infected w¹¹¹⁸ flies. (B) PCR was performed using a forward primer for pUAST-attB and a reverse primer for lncRNA-CR11538. (C) The lncRNA-CR11538 expression level was detected in the CR11538-overexpressing flies, normalized to their control levels. Minimum free energy method (D) and thermodynamic ensemble method (E) were used to predict its secondary structure on RNAfold Webserver. For all tests, p value < 0.05 was considered as statistically significant. *** p < 0.001 vs. the control groups.

Figure 2

Enrichment analysis of DEGs in the CR11538-overexpressing flies after stimulation with M. luteus. (A) Volcano map showing DEGs among Gal80^ts; Tub> CR11538 and Gal80^ts; Tub-Gal4/P{CaryP}attP40 after M. luteus challenge. Red: upregulated genes (>2-fold difference in relative expression and adjusted p < 0.05); blue: downregulated genes (<−2-fold difference in relative expression and adjusted p < 0.05). (B) Biological process enrichment analysis results were obtained using all DEGs. (C) Network diagram showing pathways and corresponding genes in KEGG pathway enriched with all DEGs. “size” represents the number of differentially expressed genes (DEGs) contained in each pathway. (D) GSEA analysis on the RNA-seq data between the CR11538-overexpressing flies and the control. The GSEA diagram is divided into three parts. The first part is the line graph of gene Enrichment Score. The second part uses lines to mark the genes under the gene set. The third part is the rank value distribution map of all genes.

Figure 3

lncRNA-CR11538 inhibits Toll signaling pathway in vivo. Drs (A) and Mtk (B) expression levels in the CR11538-overexpressing flies at different time points (0 h, 6 h, 12 h, 24 h) after M. luteus infection. (C) Changes in the survival rate of the CR11538-overexpressing flies and the control flies treated with PBS or E. faecalis. Measurements made after 36 h. Gal80^ts; Tub-Gal4/P{CaryP}attP40-PBS (n = 115), Gal80^ts; Tub > CR11538-PBS (n = 118), Gal80^ts; Tub-Gal4/P{CaryP}attP40-E. faecalis (n = 108), Gal80^ts; Tub > CR11538-E. faecalis (n = 109). For all tests, p value < 0.05 was considered as statistically significant. ** p < 0.01, *** p < 0.001 and ns, no significance vs. the control groups.

Figure 4

LncRNA-CR11538 interacts with Dif/Dorsal. (A) U6 and GAPDH are marker genes used to confirm partitioning between nucleus and cytoplasm. (B) LncRNA-CR11538 expression in nucleus and cytoplasm detected by RT-qPCR. Predicted scores for interaction potential of lncRNA-CR11538 with Dif or Dorsal via IncPro (C) and RPISeq (D) databases. LncRNA-CR11538 fold enrichment measured by RIP-qPCR using anti-V5 antibody to immunoprecipitate overexpressed Dorsal-V5 (E) and Dif-V5 (F) in S2 cells. For all tests, p value < 0.05 was considered as statistically significant. *** p < 0.001 vs. the control groups.

Figure 5

LncRNA-CR11538 prevents Dif/Dorsal from binding to AMP promoters and inhibits their transcriptions. ChIP-qPCR was performed on lncRNA-CR11538 overexpressing S2 cells with Dif-V5 (A) and Dorsal-V5 (C) overexpression normalized to control expression levels. The dual luciferase reporter assay detected transcriptional activity of Dif (B) and Dorsal (D) with or without lncRNA-CR11538 overexpressing on Drs and Mtk promoters. For all tests, p value < 0.05 was considered as statistically significant. * p < 0.05; ** p < 0.01 and *** p < 0.001 vs. the control groups.

Figure 6

LncRNA-CR11538 facilitates immune homeostasis recovery. The dynamic expression levels of Drs (A), Dif (B), Dorsal (C), lncRNA-CR11538 (D) in the wild-type Drosophila infected with M. luteus were detected by qRT-PCR at 0 h, 3 h, 6 h, 12 h, 24 h and 48 h after stimulation. For all tests, p value < 0.05 was considered as statistically significant. * p < 0.05; ** p < 0.01 and *** p < 0.001 vs. the control groups.

Figure 7

Schematic illustration of the mechanism by which lncRNA-CR11538 facilitates immune homeostasis recovery. Left diagram: In the early stage of immune response, Gram-positive bacteria activate Toll signaling pathway and promote Dif/Dorsal entering the nucleus to enhance AMP transcriptions, thereby eliminating pathogens. Right diagram: In the late stage of immune response, redundant AMPs increase the expression of lncRNA-CR11538, which acts as a Dif/Dorsal decoy to sequester them away from AMP promoters and inhibits the transcriptions of AMPs to restore immune homeostasis.