The Lifespan of D. melanogaster Depends on the Function of the Gagr Gene, a Domesticated gag Gene of Drosophila LTR Retrotransposons

Simple Summary

Transposable elements serve as a potent genetic resource for the host genome, playing a key role in the formation of diverse regulatory sequences and new genes. The evolutionary process of adaptation of transposable element sequences by a host for its own benefit is termed ‘molecular domestication’. Among genetic model organisms, Drosophila melanogaster is extensively used for studying LTR retrotransposons, a class I of transposable elements present in diverse groups within its genome. Nonetheless, the molecular domestication of LTR retrotransposons in D. melanogaster remains underexplored. Our study focuses on the role of the domesticated LTR retrotransposon capsid gag gene, Gagr, in the D. melanogaster genome. We conducted a comparative analysis of flies with a Gagr gene knockdown in all tissues against control flies through physiological testing and RNA-sequencing experiments. The flies with the Gagr gene knockdown demonstrated a reduced lifespan compared to control flies. At the same time, flies with the Gagr gene knockdown exhibited altered transcription patterns in categories of genes related to developmental control, morphogenesis, and central nervous system functionality. Our findings highlight the crucial role of the Gagr gene in maintaining immune response and homeostasis.

Abstract

(1) Background: The Gagr gene in Drosophila melanogaster’s genome originated from the molecular domestication of retrotransposons and retroviruses’ gag gene. In all Drosophila species, the Gagr protein homologs exhibit a conserved structure, indicative of a vital role. Previous studies have suggested a potential link between the Gagr gene function and stress responses. (2) Methods: We compared flies with Gagr gene knockdown in all tissues to control flies in physiological tests and RNA-sequencing experiments. (3) Results: Flies with the Gagr gene knockdown exhibited shorter lifespans compared to control flies. Transcriptome analysis revealed that Gagr knockdown flies showed elevated transcription levels of immune response genes. We used ammonium persulfate, a potent stress inducer, to elicit a stress response. In control flies, ammonium persulfate activated the Toll, JAK/STAT, and JNK/MAPK signaling pathways. In contrast, flies with the Gagr gene knockdown displayed reduced expression of stress response genes. Gene ontology enrichment analysis identified categories of genes upregulated under ammonium persulfate stress in control flies but not in Gagr knockdown flies. These genes are involved in developmental control, morphogenesis, and central nervous system function. (4) Conclusion: Our findings indicate the significance of the Gagr gene in maintaining immune response and homeostasis.

1. Introduction

Molecular domestication of retroelements, including retrotransposons and retroviruses, is a significant factor in forming new genes in eukaryotic genomes. All three genes of retroelements with long terminal repeats (LTRs)—gag, pol, and env—domesticated homologs with functions beneficial to the host organism have been identified. The capsid gene, gag, exhibits the greatest diversity in such genes. In mammals, gag domestication is exemplified by gene families like PNMA (ParaNeoplastic Ma Antigens), MART (Mammalian RetroTransposons), and SIRH (Sushi-Ichi Retrotransposon Homologues) [1]. The PNMA family includes genes that regulate apoptosis [2], while many MART/SIRH family genes are expressed in the placenta, playing vital roles in its early formation and development [3]. Another notable example of gag domestication is the SCAN domain, which is prevalent among Tetrapoda transcription factors and influences various biological processes such as embryonic development, hematopoiesis, and metabolism [4]. Additionally, several domesticated gag genes contribute to retrovirus protection, as observed in mice with genes like Fv1, Fv4, Rmcf1, and Rmcf2 [5]. Recent characterizations of domesticated retroelement sequences include the LINE retroelement upstream of the Pparg gene, essential for adipogenesis [6], and the PRLH1 transcript from the endogenous retrovirus ERV-9, involved in repairing double-strand breaks [7].

The Drosophila melanogaster Gagr gene serves as an invertebrate example of the molecular domestication of the gag gene from retrotransposons/retroviruses [8]. In all sequenced Drosophila genomes, Gagr genes possess a highly conserved structure, reflecting long-term domestication under stabilizing selection [8]. The function of the Gagr gene remains unknown.

Studies suggest a potential link between the Gagr gene and immune responses or stress-related processes [9]. Gagr gene research indicates its involvement in several crucial processes associated with stress reactions. For example, bacterial lipopolysaccharides induce Gagr expression in S2 cells, dependent on MAPK/JNK stress signaling pathway regulators Tak1, hep, and bsk [10]. Additionally, intrabdominal injection of viruses like DCV, FHV, and SINV significantly increases Gagr expression [11]. We previously identified a binding motif for the kayak transcription factor, a component of the JNK signaling pathway, and two motifs for the Stat92E transcription factor, part of the JAK/STAT pathway, in the Gagr gene promoter [9].

Protein-protein interactions of Gagr were established in D. melanogaster S2R+ cells [12]. The Gagr protein interacts with five proteins (CG3687, CG6013, 14-3-3e, Pdi, and eIF3j); most of them have stress-related functions [8]. 14-3-3e regulates MAPK and other stress pathways [13]; Pdi is crucial in endoplasmic reticulum stress and the unfolded protein response [14]; eIF3j is necessary for IRES-dependent translation during cell stress [15]. The CG3687 gene, less studied in D. melanogaster, is associated with a flightless phenotype when knocked down [16]. The function of the CG6013 protein, homologous to the human CCDC124 protein, remains unknown, but in Saccharomyces pombe, the orthologous Oxs1 gene product is a cofactor in the Pap1/Oxs1 signaling pathway [17]. Therefore, investigating the Gagr gene’s role in cell stress, given its activation under stress and protein interactions, is crucial.

To study the Gagr gene’s function, we employed the reduction of Gagr transcription through RNA interference-induced knockdown. The flies with Gagr gene knockdown in all tissues were tested for their lifespan, motility, and transcriptomic responses to ammonium persulfate (APS), a cellular homeostasis disruptor.

2. Materials and Methods

2.1. Drosophila Melanogaster Strains and Cultivation Conditions

The following strains of D. melanogaster were used: w¹¹¹⁸; tub-GAL4, driver strain from Bloomington Drosophila Stock Center (y¹, w¹¹¹⁸; P{w^+mC = tubP-GAL4}LL7 P{ry^+t7.2 = neoFRT}82B/TM6B, Tb¹); P{KK109908}VIE-260B, KK RNAi strain from the Vienna Drosophila Resource Center, carrying a transgenic construct for expression of dsRNA for the Gagr gene RNA interference under UAS region control. Fly stocks were maintained in a standard nutrient agar medium at 25 °C. To induce interference, females of the UAS-Gagr RNAi strain were crossed with males of the tub-GAL4 driver strain. Thus, analyzed hybrids with knockdown of the Gagr gene, which we called Gagr^RNAi, were obtained. Females of the w¹¹¹⁸ strain were crossed with tub-GAL4 males as a control.

2.2. Physiological Tests

Lifespan was measured at 27 °C on the standard medium and on the medium supplemented with 0.1 M APS. To analyze lifespan flies at the age of 1 day were selected, separated by sex, and put into separate test tubes of 20–30 flies. The number of individuals in the test tube was checked every 2–3 days (or hours—for APS medium); the food was replaced every 5 days. To measure the motility of the imago, the climbing test was used [18] with modifications: 30 adults were placed in an empty long tube 17 cm long. Flies were dropped to the bottom of the test tube by mechanical tapping. Next, the time it took for each fly to achieve its maximum vertical climb rate was measured. Two independent repeats were performed.

2.3. RNA Extraction and RT-PCR

Total RNA was isolated from the Gagr^RNAi and control females and males (5–7 days old) after standard cultivation or after 24-h exposure to 0.1 M APS. RNA was isolated from pools (five females or seven males) in 5–7 biological replicates using the ExtractRNA reagent (Evrogen, Moscow, Russia), according to the manufacturer’s protocol; then, it was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was carried out using an MMLV-RT kit (Evrogen), according to the manufacturer’s protocol, with random primers (Evrogen). For quantitative PCR with the obtained cDNA, a Taq polymerase-based reaction mixture with SYBR Green I (Evrogen) was used in accordance with the manufacturer’s protocol. The reaction was performed using a Mini Opticon Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The relative expression of the genes Gagr, TotA, TotC, AttB, CecA2, Socs16D, Spn88Eb, CG33346, Nazo, Ppo1, Spn28Dc, CG1304, CG10232, Ser6, CG10051, normalized to the expression of two reference genes, Tub84D and EloB, was analyzed. Amplification was performed with primers shown in Table S3. A histogram was constructed in the GraphPad Prism 9 program (https://www.graphpad.com/) to present the expression results. Statistical significance was assessed using the nonparametric Mann–Whitney test in GraphPad Prism 9.

2.4. RNA-Sequencing and Data Processing

RNA was isolated from pools (five females or seven males) in 5–7 biological replicates using the ExtractRNA reagent (Evrogen), according to the manufacturer’s protocol, and then it was treated with DNase I (Thermo Fisher Scientific). RNA concentration and integrity were evaluated by a fluorimetric assay with Qubit 4 (Thermo Fisher Scientific) and capillary electrophoresis on Tape Station (Agilent Technologies, Santa Clara, CA, USA), respectively. All samples were prepared in one experiment (3 repeats for each sample). Strand-specific libraries were prepared by the NEB Next Ultra II Directional RNA Library Preparation kit (NEB, Ipswich, MA, USA) and sequenced (100 nucleotides, single end) with a median depth of 25 million reads per sample by NovaSeq 6000 (Illumina, San Diego, CA, USA). Low-quality reads, and adapter sequences were deleted (Timmomatic tool, v0.36), then the reads were aligned to the BDGP6 primary genome assembly. Uniquely aligned reads were counted for known exons of each gene using the R package (R environment). For the reference genes ppl, Tbp, Gapdh1, tub, RPL40, and SdhA, the expression deviation for each gene in the sample was assessed and normalized to the expression value of the corresponding gene in the control samples without exposure to APS. Deviations for different genes averaged +/−0.3. Differential expression analysis was performed by the DESeq2 package (version 1.41.0) (https://bioconductor.org/packages/release/bioc/html/DESeq2.html (accessed on 30 April 2023)). The heatmap of the differentially expressed genes is done using the heatmap package (https://www.bioinformatics.com.cn/plot_basic_cluster_heatmap_plot_024_en (accessed on 3 October 2023); [19]). Differentially expressed genes (DEGs) were protein-coding genes with |Log2 Fold Change| ≥ 0.6, Padj < 0.05. Metascape analysis tools were used to identify functional enrichment categories of DEGs (http://metascape.org (accessed on 3 October 2023); [20]). The Gene Ontology Resource (https://geneontology.org/ (accessed on 3 October 2023)) was used to search for molecular function enrichment. To determine tissues associated with the transcriptome response, we used data from the FlyAtlas 2 project [21]. We assessed correlations of tissue-specific expression values for DEGs and built a gene co-expression network for correlations of 0.8 or more using Cytoscape analysis based on the graph-oriented clustering method MCODE (https://cytoscape.org/ (accessed on 3 October 2023)).

3. Results

3.1. Physiological Tests of the Gagr Gene Knockdown Flies

3.1.1. Knockdown of the Gagr Gene Does Not Affect Embryonic and Larval Viability of Flies

To initiate the Gagr gene RNA interference, we crossed females from the VDRC KK strain P{KK109908}VIE-260B, which carry a transgenic construct for dsRNA expression of the Gagr gene fragment under UAS region control, with males from the tub-GAL4 driver strain. For controls, females of the w¹¹¹⁸ strain were crossed with tub-GAL4 males. The tub-GAL4 driver strain is heterozygous for the dominant Tubby mutation, located on the third chromosome and characterized by a short body phenotype and extra macrochetes on both sides of the head. Opposite the Tubby allele, on the homological chromosome, the GAL4 gene is located, which is essential for RNA interference induction. The genetic construct enabling Gagr gene knockdown is located on the second chromosome of the responder strain. Crossbreeding of the driver and responder strains should result in offspring with a 1:1 ratio of long-bodied knockdown and short-bodied flies as a byproduct. To validate the 1:1 hypothesis, we employed the chi-square (χ²) method (

Table 1. Number of imago in crosses of the Tub-GAL4 driver with the w¹¹¹⁸ and VDRC P{KK109908}VIE-260B strains.

Strain		Sum of Flies	Flies with a Short Body			Flies with a Long Body
			Males	Females	Sum	Males	Females	Sum
tub-GAL4 × w1118	Observed	897	224	251	475	193	229	422
	Expected		224.25	224.25	448.5	224.25	224.25	448.5
tub-GAL4 × P{KK109908}VIE-260B	Observed	1351	315	395	710	255	386	641
	Expected		337.75	337.75	675.5	337.75	337.75	675.5

). For the control hybrids, χ² = 3.14, α = 0.076. Similarly, in the Gagr knockdown hybrids, χ² = 3.52, α = 0.061. Therefore, the knockdown of the Gagr gene does not influence the embryonic and larval viability of the flies.

3.1.2. Knockdown of the Gagr Gene Affects the Lifespan of Flies under Standard and Stress Conditions

We investigated the lifespan of Gagr knockdown flies (hereafter referred to as Gagr^RNAi) compared to control flies (Figure 1A). The maximum lifespan observed for Gagr^RNAi males was 45 days, while control males reached up to 55 days. Similarly, Gagr^RNAi females had a maximum lifespan of 55 days compared to 75 days for control females. Consequently, Gagr^RNAi flies exhibited a reduced maximum lifespan compared to the control group.

Subsequently, we assessed the survival rate of flies on a medium containing 0.1 M APS. Under these conditions, the maximum lifespan for Gagr^RNAi males was 30 h, and for females, it was 50 h (Figure 1B). In comparison, control males and females had maximum lifespans of approximately 50 h and 64 h, respectively. Therefore, under APS-induced stress, Gagr^RNAi flies (both males and females) demonstrated a decreased survival rate compared to controls. Notably, the females showed greater resilience to APS stress than males.

3.1.3. Knockdown of the Gagr Gene Does Not Lead to Changes in Adult Motility

Flybase data indicated that the knockdown of the CG3687 gene, which encodes a protein interacting with the Gagr protein, results in a flightless phenotype (refer to Flybase report FBgn0034097). We hypothesized that the knockdown of the Gagr gene might impact the function of the CG3687 protein, thereby affecting the motility of flies. However, our experiments revealed no significant difference in vertical ascent time between the Gagr gene knockdown flies and control flies. All tested individuals were able to cover a distance of 17 cm in approximately 10 ± 1 s. Therefore, our findings suggest that the knockdown of the Gagr gene does not affect fly motility.

3.1.4. Knockdown of the Gagr Gene in Females Promotes the Occurrence of Melanin Capsules in the Fat Body

Although no morphological changes were evident in the overall appearance due to the Gagr gene knockdown, we observed that only the females, not the males, developed black spots visible through the abdominal cuticle. Upon dissection, these spots were identified as multiple black granules, or melanin capsules, within the fat body (Figure 1C). Interestingly, similar phenomena were not observed in males with the Gagr gene knockdown.

3.2. Transcriptomic Analysis of the Gagr Gene Knockdown Flies

3.2.1. Differentially Expressed Genes in the Gagr Knockdown Flies during Normal and Stress Conditions

We conducted a comparative analysis of the transcriptomes of control and Gagr^RNAi adult flies under both normal and APS stress conditions (Table S1). For eight datasets (control females, control males, Gagr^RNAi females, Gagr^RNAi males, each under both stress and normal conditions), we constructed a heatmap of average gene expression, as depicted in Figure 2A. This heatmap clearly demonstrates that the expression patterns of identified DEGs can effectively distinguish between the four types of samples for both females and males. In females, the transcriptomes of control and Gagr^RNAi flies are similar under both normal and APS stress conditions (Figure 2A). In males, however, the transcriptomes are clustered not by the presence or absence of the Gagr gene but rather by the environmental conditions, suggesting that Gagr knockdown influences the male transcriptomic response to APS stress.

Given the absence of a universally accepted fold change threshold for defining DEGs due to the variation in transcription rates among genes, we opted to set an initial threshold at 1.5. Under normal conditions, among approximately 13.5 thousand genes, 509 exhibited a change in transcription level of 1.5-fold or more (|Log2FoldChange(LFC)| > 0.6, Padj < 0.05) in females, and 346 in males with Gagr gene knockdown. Specifically, in Gagr^RNAi females, 297 genes showed increased transcription levels compared to control females, while in males, 191 genes were upregulated. Conversely, the transcription of 263 genes was downregulated in Gagr^RNAi females relative to controls and 153 genes in Gagr^RNAi males. Notably, 34 genes showed increased transcription in both sexes, including genes for antimicrobial peptides Attacins and Drosomicins, regulated by the Imd and Toll signaling pathways. Similarly, the transcription of 28 genes was reduced in both sexes, comprising genes such as Lsp1beta, MtnE, Cyp4d1, Yp1, and Mal-B1, which are involved in metabolic processes. These changes in gene expression may account for the observed alterations in lifespan in both females and males.

Upon APS exposure, we noted significant transcriptional changes in a large number of genes in both sexes (Table S1). In control females, 418 genes showed increased expression, while 795 exhibited decreased expression (|LFC| > 0.6, Padj < 0.05). In Gagr^RNAi females, 411 genes increased, and 458 genes decreased their expression. Among control males, 171 genes increased, and 44 genes decreased their expression, while in Gagr^RNAi males, 67 genes showed increased expression, and 42 genes showed decreased expression.

To establish the threshold for assessing DEGs, we conducted RT-PCR verification of RNA-seq data for the Gagr gene and 14 DEGs related to immune response (Figure 2B). The PCR analysis encompassed DEGs in both control and Gagr^RNAi flies, including the Gagr gene itself; antimicrobial peptide genes TotA, TotC, AttB, and CecA2, highly expressed in the fat body; the Socs16D gene, a repressor of the JNK/MAPK pathway; the Nazo gene, an antiviral effector of the Imd pathway; serine endopeptidase inhibitor genes Spn88Eb and Spn28Dc, involved in regeneration and melanization inhibition; serine endopeptidase genes Ser6, CG1304, and CG10232; the metalloexopeptidase gene CG10051; the apoptotic endopeptidase gene G33346; and Ppo1, a major gene for prophenoloxidase involved in melanization.

These genes were selected based on their differential expression levels in males and females, with LFC values greater than 0.6 and varied p-values, including some with |LFC| > 0.6 and p-value > 0.05. Since the cDNA library and RNA-seq data were derived from a single experiment, we performed verification for four groups: control and Gagr^RNAi males and females, analyzing 60 samples in total. PCR for each sample was conducted in 5–7 biological replicates.

In most instances, our PCR results corroborated the RNA-seq data, demonstrating good agreement between RNA-seq p-values and the reliability of PCR findings. Notably, the accuracy of expression level determination was influenced by gene expression levels (high or low) and the variability of expression. Genes like CecA2 (males), Nazo, and CG10051 showed differential expression in RNA-seq but had non-significant p-values. We encountered only two discrepancies between PCR and RNA-seq results: in females, PCR analysis did not confirm RNA-seq data for the TotA and CecA2 genes. These inconsistencies might be attributed to experimental errors due to limited gene coverage or slight variances in gene expression between younger and older females used in the study (ages 5–7 days).

For enrichment analysis, we set the thresholds at |LFC| > 1 and Padj < 0.05. Raising the threshold did not alter the enrichment; however, it eliminated categories that exhibited low levels of significance. Functional category enrichment analysis (Gene Ontology, GO and KEGG pathways) indicated a significant increase in transcription of genes responsive to Gram-positive and Gram-negative bacteria, genes of the Toll and Imd signaling pathways, and cellular heat response in Gagr^RNAi females (Figure 3A). In males, fewer functional categories were enriched, with immune response genes being the most prominent.

Low-expressed genes in Gagr^RNAi females were enriched in metabolic processes, localization, and biological processes related to interspecies interaction. In Gagr^RNAi males, low-expressed genes were enriched in response to biotic stimulus and lipid metabolic processes. Therefore, the most represented functional category of genes in both males and females, distinguishing Gagr^RNAi from control flies, was that of immune response genes, including 34 DEGs in females and 19 in males.

Under APS stress conditions, genes that increased their expression in control females showed greater enrichment in functional gene categories than in control males and Gagr^RNAi females (Figure 3B). Many of these categories are associated with development, including central nervous system development. This indicates that the transcriptomic response to APS is sex-specific and less pronounced in males. The knockdown of the Gagr gene significantly alters the response in females.

In our study of the tissue-specific response in control females, which exhibited a significant reaction to APS, we observed a systemic response encompassing numerous tissues (Figure 4). Specifically, in control females, we identified that the highest number of genes with increased expression in response to APS were predominantly active in the central nervous system and the digestive system. Conversely, the genes exhibiting the most substantial decrease in expression were those primarily active in the gut, endocrine system, and reproductive system.

3.2.2. Some Genes Are Not Induced by APS Stress in Flies with the Gagr Gene Knockdown

We next focused on identifying genes that were not activated in Gagr^RNAi females but showed increased expression in control females in response to stress, specifically those with a LFC greater than 1.5 for controls and less than 0.5 for Gagr^RNAi. A total of 195 such genes were identified. Functional category enrichment analysis revealed that these genes are predominantly associated with developmental processes (Figure 5A).

Subsequently, using the MetaScape tool, we performed ontology cluster enrichment analysis for the genes overexpressed in control females (Figure 5B). This analysis involved converting a subset of representative terms into a network layout. In this network, each term is depicted as a circle node, with the node’s size indicating the number of input genes associated with that term and the color representing its cluster identity (nodes with the same color belong to the same cluster). Terms sharing a similarity score greater than 0.3 are connected by edges, where the thickness of each edge corresponds to the similarity score. This network was visualized using Cytoscape with a “force-directed” layout and edge bundling for enhanced clarity. Notably, all clusters in this network are interconnected.

The enrichment of functional categories indicated that genes activated in control females but not in Gagr^RNAi females show tissue specificity (Figure 5C). These genes are particularly associated with expression in the crop, midgut, hindgut, and central nervous system. Interestingly, the tissue-specific expression pattern of these genes closely mirrors that of the Gagr gene itself, as per the expression data available in FlyBase (Figure 5D).

In our study, we focused on identifying genes that were activated by stress in control females but remained inactive in Gagr^RNAi females. Functional enrichment analysis of this gene set, based on molecular function, revealed a distinct group of 19 transcription factors (Fold Enrichment: 4.76; p-value = 8.51 × 10⁻⁹, FDR = 2.53 × 10⁻⁵). These transcription factors include run, ss, ase, sr, Antp, Sox21a, esg, grh, ham, Dfd, ich, nerfin-1, dmrt99B, grn, Kr-h1, acj6, rib, and tap (

Table 2. Genes not activated by stress in the Gagr^RNAi females.

Gene	Biological Function of the Protein (According to FlyBase)
run	Contributes to axon guidance, dendrite morphogenesis, and germ-band extension
ss	Plays a key role in defining the distal regions of the antenna and the legs
ase	Acts together with other proneural genes in nervous system development, which involves N-mediated lateral inhibition
sr	Induces the fate of tendon cells in the embryo as well as in the adult fly
Antp	Takes a part in a developmental regulatory system that specifies segmental identity in the pro- and mesothorax
Sox21a	Involved in the differentiation of stem cells in the midgut
esg	Contributes to stem cell maintenance, tracheal morphogenesis, and neuroblast differentiation
grh	Responsible for the proper expression of many genes primarily involved in epithelial cell fate, barrier formation, wound healing, tube morphogenesis, and proliferation of larval neuroblasts
ham	Regulates neuron fate selection in the peripheral nervous system and olfactory receptor neurons
Dfd	Involved in proper morphological identity of the maxillary segment and the posterior half of the mandibular segment
ich	Regulates the transcription of factors involved in the formation of a mature apical extracellular matrix, which is essential for the integrity and shape of seamless tubes
nerfin-1	Regulates early axon guidance at the embryonic stage and is required for the maintenance of larval neuron differentiation
dmrt99B	Involved in sex differentiation
grn	Regulates the expression of receptors and adhesion molecules involved in axon guidance
Kr-h1	Involved in axon pathfinding, neurite, and axon remodeling, as well as pupal photoreceptor maturation
acj6	Acts in odor receptor gene expression and axon targeting of olfactory neurons
rib	Required for development of the salivary gland and trachea, as well as for dorsal closure
tap	May play a role in the specification of the sugar-sensitive adult gustatory neuron

). The primary biological functions of these genes’ products are associated with the development and functioning of the nervous system. Therefore, it appears that in flies with a knockdown of the Gagr gene, the disrupted expression of many genes may be linked to impaired activation of these transcription factors. This suggests that their activation is dependent on the Gagr gene.

3.2.3. Transcription of Signaling Pathways Genes Is Disrupted in the Flies with the Gagr Gene Knockdown

Our functional enrichment analysis for genes with increased expression revealed numerous terms associated with stress response. Consequently, we specifically examined how APS influences the expression of genes involved in major stress signaling pathways.

Initially, we evaluated the impact of APS on the JNK/MAPK and JAK/STAT stress cascades. In control females, APS was found to elevate the expression of key transcription factors of the JNK cascade, specifically jra (Jun) and kay (Fos) (Figure 6A). However, the key kinases of the JNK cascade did not show regulation at the transcriptional level. Other JNK cascade components that were activated at the gene expression level included Gadd45, which is linked to the regulation of the localization of JNK cascade proteins, and raw, a gene encoding a membrane protein involved in dendrite patterning and the subcellular localization of JNK signaling components. Additionally, the puc gene, encoding a serine/threonine protein phosphatase that forms a negative feedback loop in the JNK cascade, was also activated [22]. The genes Pvf2 and Pvr, acting as a ligand and receptor, respectively, for activating the MAPK cascade, and the MAP kinase p38c gene, involved in stress and wound responses, also showed increased expression in response to APS [22,23]. The expression of JNK target genes Dpp, Mmp1, and wg, essential for cellular processes such as apoptosis and cell proliferation, was elevated as well [24]. Furthermore, the JNK pathway promotes the activity of the Foxo transcription factor gene, which in turn activates the expression of cytoprotective genes like Fas1,2,3, GADD45, and Thor [25]; these were found to be upregulated in control flies in response to APS (Figure 6A).

Therefore, it appears that the JNK cascade can be regulated at the level of expression of its components in response to APS. This regulation primarily involves the activation of the expression of extracellular ligands, their receptors, and transcription factors, but not the main kinases.

The transcriptional response to APS in Gagr^RNAi males and females was notably subdued. This suggests that the knockdown of Gagr disrupts the activation of gene expression involved in the JNK cascade.

In our analysis of the JAK/STAT signaling pathway response to APS (Figure 6B), we observed activation in the expression of certain genes in both control females and males: upd2 (a ligand of the JAK/STAT cascade) and Socs36E (a negative regulator of the JAK/STAT cascade). However, the transcriptional activation of the STAT92E transcription factor was exclusively noted in control females. Therefore, the JAK/STAT cascade regulation, in response to APS, occurs at the transcription level of its components. This regulation involves the activation of cytokines and a negative regulator of the cascade. The knockdown of Gagr led to reduced activation of STAT92E gene expression in females and upd2 and upd3 in males but did not affect the Socs36E gene in either sex or upd2 in females.

We also investigated the NFkB signaling pathways, Toll and Imd (Figure 6C). While their role in the innate immunity of Drosophila is well-documented, their protective function under abiotic stress is less understood. In control females and males, we detected no significant changes in the regulation of Imd-signaling components’ expression in response to APS, except for a modest but statistically significant increase in the transcription of the genes ken in females and Dredd in males.

For the Toll signaling pathway, however, we observed changes in the expression of several secreted factors (ligands, proteases, etc.) that positively regulate Toll signaling activity: an increase in the expression of the GNBP2 gene, encoding the Gram-negative bacteria binding protein, and the genes spz4 and spz6, involved in Toll pathway-dependent AMPs production; a decrease in the expression of the GNBP3 gene, another Gram-negative bacteria binding protein gene, and the genes PGRP-SC1a, PGRP-SC2, and PGRP-SD, encoding peptidoglycan recognition proteins, and the SPE gene, coding a protease responsible for cleaving the Toll ligand. Control males also showed a decrease in the expression of several genes in response to APS.

The activation of intracellular Toll signaling components in response to APS was not detected in Gagr^RNAi males and females, indicating that the knockdown of Gagr disrupts the activation of gene expression involved in NfkB signaling pathways.

Furthermore, in control females, we noted an increased transcription level of genes activated by ER and oxidative stresses, regulated by the transcription factors Hsf1 (genes of the Hsp70 family) and Xrp1 (genes of the GstD family) (

Table 3. Genes of the GstD family and Hsp70B family activated by APS stress in the control but not in the Gagr^RNAi females.

Genes	Control Females	GagrRNAi Females
GstD2	LFC = 2.2, Padj = 0.01	Not activated
GstD5	LFC = 2.1, Padj = 0.00001	Not activated
GstD8	LFC = 1.6, Padj = 0.006	Not activated
Hsp70Ba	LFC = 3.7, Padj = 0.04	Not activated
Hsp79Bb	LFC = 3.4, Padj = 0.0000003	Not activated
Hsp70Bc	LFC = 3.3, Padj = 0.002	Not activated

). Therefore, the knockdown of Gagr also disrupts the activation of gene expression involved in the ER stress response.

4. Discussion

We observed that flies with a knockdown of the Gagr gene had a shorter lifespan than control flies. Additionally, the Gagr gene knockdown in females led to the formation of melanin capsules in the fat body. Some of the phenotypes of Gagr knockdown may be explained by the observed changes in gene expression. The most prominently represented functional category of genes in both sexes, differentiating Gagr^RNAi from control flies, is related to immune response, particularly the genes of antimicrobial peptides (AMPs).

The impact of AMP gene overexpression on the lifespan of Drosophila is a subject of mixed findings. Some studies have shown that AMP overexpression, including Drosocin and Cecropin A1, significantly extends lifespan [26], with such flies displaying reduced immune pathway activity, lesser intestinal regenerative processes, lower stress response, and delayed degradation of gut barrier integrity [27]. Conversely, other research suggests that AMP overexpression can contribute to aging through cytotoxic effects in Drosophila tissues [26], as chronic immune response activation may cause collateral damage to host tissues, potentially leading to premature aging and age-related diseases [28,29,30].

AMP expression is regulated by NFkB family members, including transcription factors Dif, Relish, and Dorsal [31]. Additionally, subsets of AMPs can be directly activated by the transcription factor Foxo, depending on the metabolic status of the fly, illustrating cross-regulation between metabolism and innate immunity [32]. In the midgut, AMP expression is controlled by the negative transcription regulator caudal [33,34]. However, we observed no significant changes in the gene expression of caudal and foxo transcription factors. Our data indicate activation of Toll and Imd signaling pathways in Gagr^RNAi flies. Consistent with the heightened activity of AMP genes, both Gagr^RNAi males and females exhibit shorter lifespans than control flies.

Beyond the activation of AMP genes, we observed melanotic nodules in females with the Gagr gene knockdown, potentially indicating autoimmune reaction induction. Of the Ppo prophenoloxidase family genes, only Ppo1 showed statistically significant transcriptional changes: Ppo1 expression is lower in females than in males for both knockdown and control flies, and its expression in Gagr^RNAi flies is further reduced under stress conditions. FlyBase data reveals that Ppo1 is highly expressed in muscle cells and carcass, with overall higher expression in males than females.

Prophenoloxidase activation is partly regulated by the serine protease inhibitor Spn27A. Spn27A mutant larvae display melanotic phenotypes and excessive melanization in response to immune challenges [35] linked to Toll pathway activation [36,37]. Constitutive pathway activation, as in Toll gain-of-function or cactus loss-of-function mutants, leads to hemocyte overproliferation, especially lamellocytes, resulting in melanotic nodule formation [38].

Other signaling pathways can also activate melanization. Immune challenges in larvae with constitutive PGRP-LE expression upstream of the Imd pathway led to melanotic masses in the cuticle and hemolymph [39]. Activation of pathways like Ras/MAPK in hemocytes induces hemocyte proliferation and melanotic mass formation [40,41]. Constitutive JAK/STAT signaling activation, as in the dominant Jak mutation hopTum-l, induces TotA gene upregulation, plasmatocyte overproliferation, and lamellocyte differentiation, leading to melanotic masses in larvae and adult flies [42]. The tuSz1 mutant shows a temperature-sensitive self-encapsulation phenotype directed at its own posterior fat body tissue [43,44], possibly due to a gain-of-function mutation in the hop gene and a loss-of-function mutation in the GCS1 gene, disrupting the N-glycosylation pathway in the posterior fat body [45]. This demonstrates that N-glycosylated extracellular matrix proteins act as self-associated molecular patterns (SAMPs), with activated innate immune cells attacking tissues lacking these SAMPs. The self-tolerance mechanism may also initiate immunity through “missing-self recognition” if pathogens lack a self-signal on their surface [46].

Notably, GCS1 gene transcription in Gagr^RNAi females and males does not change significantly, precluding a direct association of melanization with GCS1 function in females. However, under stress conditions, GCS1 expression significantly decreases in Gagr^RNAi females (LFC = −0.59, Padj = 0.0001), indicating disrupted regulation of this gene.

Our previous research has shown that transcription of the Gagr gene is most notably induced in females by the strong oxidant ammonium persulfate [9]. APS primarily affects membrane proteins on the cell surface, often leading to decreased cell viability and increased apoptosis [47] and causes significant oxidative stress in lysosomes, inducing epithelial-mesenchymal transition via lysosomal oxidative stress [48]. Hence, APS triggers a robust stress response, yet transcriptomic studies of APS’s effects on D. melanogaster are limited.

We found that APS significantly alters the female transcriptome, activating genes associated with protective stress responses (immune response, inflammation, chitin metabolism) and suppressing genes involved in fat, protein, and carbohydrate metabolism. Stress-associated signaling pathways JNK, JAK/STAT, and Toll are regulated in response to APS (Figure 7), mainly through transcription activation.

Knockdown of the Gagr gene disrupts the normal activation of stress-associated signaling cascades, including JNK, JAK/STAT, and Toll. We noted that genes activated in control flies but not in Gagr^RNAi flies are associated with activities in the digestive and central nervous systems. Interestingly, the tissue specificity of the control response to APS correlates with the tissue-specific transcription of the Gagr gene.

In response to APS stress, we observed in control females an increased transcription level of genes regulated by ER stress, specifically those under the control of transcription factors Hsf1 (genes of the Hsp70 family) and Xrp1 (genes of the GstD family) (Figure 7). This indicates that Gagr knockdown disrupts the activation of genes involved in the ER stress response.

Our findings also reveal that the transcriptomic response to stress in males is less pronounced than in females. Such sexual dimorphism in immune response is well-documented [43]. The transcription of the Gagr gene itself exhibits sexual dimorphism: it is about twice as high in males compared to females and is not induced by APS [9]. This lower gene activation in response to stress in males with Gagr knockdown could be due to a higher baseline expression of immune response genes, potentially explaining the reduced lifespan of these males under both normal and stress conditions. In control males under normal conditions, we observed increased expression of AMP genes, which might contribute to lifespan reduction, as suggested by previous studies [28,29,30].

Our data suggest that the Gagr gene is intricately integrated into the regulatory network of signaling cascades, with its transcription influenced by JNK and JAK/STAT pathway signals [9]. This is consistent with other studies where Gagr expression activation was observed in response to significant stressors like viral infection and oxidative stress. The JNK pathway plays a multifaceted role, regulating a range of processes from embryogenesis to cellular stress response. It is involved in various Drosophila and higher organisms processes, including apoptosis, proliferation, differentiation, cell migration, tumorigenesis, and regeneration [48,49]. The kayak protein, part of the AP-1 transcription factor, is involved in developmental regulation and may influence specific cell subsets in developing embryos [50]. In wounded tissues, JNK activation promotes apoptotic death in damaged cells and cellular reprogramming and proliferation in surviving cells [51].

JNK and JAK/STAT pathway activation in adult flies stimulate stem cell proliferation in response to oxidative or ER stress and infection [52]. JNK also regulates upd3 expression, an effector of the JAK/STAT pathway, crucial for optimal intestinal epithelium renewal and survival after septic injury [52]. In aging flies, widespread JNK activation in the intestinal epithelium induces excessive proliferation of intestinal stem cells (ISCs) [50]. Autophagy also plays a role in maintaining proliferation and preserving the ISC pool in Drosophila. Thus, the Gagr gene is observed in imago tissues with a high potential for stress-induced proliferative activity.

Transcriptomic analysis reveals that APS exposure triggers a systemic response involving multiple signaling pathways. However, in Gagr^RNAi flies, most signaling pathway target genes remain inactivated, indicating that Gagr knockdown leads to broad changes in gene expression, blocking stress response and post-stress tissue regeneration. This confirms the Gagr gene’s crucial role in homeostatic processes.

Considering the proven localization of the Gagr protein and its partners (CG6013, 14-3-3e, Pdi, eIF3j) associated with the ER membrane, we propose a possible scenario for the functioning of the Gagr-complex in D. melanogaster (Figure 7). Under oxidative or ER stress, Pdi, a redox-sensitive chaperone, first perceives the signal and transmits it to its partners [53]. The activity of Pdi and Gagr proteins may be modulated by phosphorylation, with 14-3-3e capable of binding to phosphorylated partners [54]. Concurrently, 14-3-3e can activate the Ras/MAPK pathway [55]. Gagr, possibly in partnership with 14-3-3e, binds to CG6013 and eIF3j, along with a set of mRNAs to the ribosome. The CG6013 protein, homologous to human CCDC124 and yeast Oxs1 proteins, may act as a transcription cofactor, similar to the role of Oxs1 in S. pombe [17].

We hypothesize that the Gagr-complex may be involved in an alternative ribosome attachment pathway to the translocon and an alternative (IRES-dependent) translation pathway under stress conditions when normal protein synthesis is hindered. This hypothesis warrants further molecular investigation.