# A dynamic Dif/lncRNA-CR42715/miR-965-3p feedback loop orchestrates toll pathway immunity response in Drosophila

## TL;DR

The study reveals a feedback loop involving a transcription factor, a long non-coding RNA, and a microRNA that controls immune responses in fruit flies.

## Contribution

A novel TF-lncRNA-miRNA feedback loop regulating Drosophila Toll pathway immunity is identified.

## Key findings

- Dif activates CR42715, which sponges miR-965-3p to enhance Dif protein synthesis and Toll signaling.
- Disruption of the CR42715/miR-965-3p feedback loop leads to dysregulated immune responses and reduced host survival.
- CR42715 acts early to boost immunity, while miR-965-3p later dampens the response, ensuring a self-limiting immune reaction.

## Abstract

The innate immune response requires precise spatiotemporal regulation for organisms to ensure effective pathogen clearance while avoiding detrimental overactivation. Although the core components of the Drosophila Toll pathway are well-established, the post-transcriptional regulatory networks, particularly those involving non-coding RNAs (ncRNAs), remain incompletely understood to date. Here, we elucidate a novel tripartite feedback loop comprising the long noncoding RNA (lncRNA) CR42715, the microRNA (miRNA) miR-965-3p, and the transcription factor (TF) Dif that dynamically modulates Drosophila Toll signaling. Firstly, our results demonstrate that upon Gram-positive bacterial challenge, Dif activates CR42715 expression, which acts as a competitive endogenous RNA (ceRNA) by sponging miR-965-3p to alleviate miR-965-3p-mediated repression of Dif and enhance Dif protein synthesis, thus facilitating Toll signaling immune responses. Secondly, disruption of this feedback loop via genetic manipulation of CR42715 or miR-965-3p leads to dysregulated AMP expression and compromised host survival. Thirdly, the temporal expression analysis reveals that CR42715 is rapidly induced early in infection to boost immunity, while miR-965-3p expression increases later, ensuring timely signal attenuation, which suggests that this dynamic Dif/CR42715/miR-965-3p feedback loop can ensure robust early-phase antimicrobial peptide production while preventing excessive late-phase immunity. Collectively, we unveil a novel TF-lncRNA-miRNA feedback loop that acts as a rheostat to ensure an effective immune response.

The innate immune system must strike a delicate balance between effective pathogen elimination and preventing excessive inflammation. How the molecular mechanism by which this balance is achieved remains incompletely understood. Here, we uncover an unprecedented temporal feedback mechanism in Drosophila that fine-tunes immune responses against Gram-positive bacteria. This feedback loop involves a dynamic interplay among the NF-κB transcription factor (Dif), the long non-coding RNA (lncRNA-CR42715), and the microRNA (miR-965-3p). We found that upon bacterial infection, Dif activates the expression of CR42715, which then functions as a molecular sponge to sequester miR-965-3p, thereby relieving its suppression on Dif mRNA. This creates a feedforward loop that amplifies early immune signaling while temporally maintaining prolonged activation through delayed miR-965-3p upregulation. Intriguingly, the expression of these components is timed. CR42715 acts early to boost immunity, while miR-965-3p increases later to dampen the immune response. This temporal feedback loop thus acts as a rheostat to ensure an effective yet self-limiting immune response. Overall, our findings reveal a previously unknown layer of regulation in a fundamental immune pathway, with potential implications for understanding similar control mechanisms in more complex organisms, including humans.