MicroRNA miR-274-5p Suppresses Found-in-Neurons Associated with Melanotic Mass Formation and Developmental Growth in Drosophila

Simple Summary In animals, including humans and flies, blood cells play crucial roles in various biological processes, such as immune response and normal development. The abnormal regulation of hematopoiesis in flies results in the formation of melanotic masses, which are also known as melanotic tumors. However, the detailed mechanisms underlying this process are not fully understood. In this study, we found that the upregulation of miR-274-5p, a small non-coding RNA, activates the JNK and JAK/STAT signaling pathways by suppressing the expression of found-in-neurons (fne) encoding an RNA-binding protein in flies. This regulation controls the formation of melanotic masses and developmental growth. Overall, our findings provide valuable insights into melanotic mass formation and developmental growth as well as the regulatory network of the JNK and JAK/STAT signaling pathways. Abstract The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. Here, we observed that the overexpression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the JNK and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly suppress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne–JNK signaling axis in melanotic mass formation and growth control.


Introduction
Blood cells play important roles in the immune response against invading pathogens and in the normal development of metazoan [1,2]. In Drosophila, hematopoiesis occurs in two different waves: in the head mesoderm of early embryos and the lymph glands of larvae [3][4][5]. During this process, Drosophila prohemocytes terminally differentiate into three types of blood cells: plasmatocytes (phagocytosis), crystal cells (melanization), and lamellocytes (encapsulation) [2,5]. Phagocytic plasmatocytes engulf apoptotic bodies and pathogens, such as bacteria and fungi, as the predominant hemocytes [6,7]. Lamellocytes, which are rare in healthy conditions, can massively differentiate after infection and form a capsule around foreign pathogens [2,8]. Melanization is facilitated by crystal cells that secrete phenol oxidase [8]. This hemocyte-mediated cellular immune response is involved in the formation of larval melanotic masses. binding protein as one of the three paralogs (Rbp9, Fne, and Elav) of the ELAV gene family, and it is primarily expressed in neuronal tissues in Drosophila [24]. According to previous reports, fne is associated with several biological processes, such as mushroom body development, male courtship performance, and synaptic plasticity [24,25]. In addition, similar to other family proteins, Fne broadly induces 3 -UTR extension in neuronal cells by blocking the use of the proximal polyadenylation site [26]. The cytoplasmic protein, Fne, undergoes a switch of cellular localization toward the nucleus due to the inclusion of a microexon encoding a nuclear localization signal under Elav-nonfunctional conditions [26]. However, functional studies on Fne have focused on its role in primarily expressed neuronal cells. As a result, other biological roles of fne in non-neuronal cells remain unknown.
In Drosophila, glia-derived miR-274 is known to play a role in regulating the growth of synaptic boutons and tracheal branches through the targeting of Sprouty [27]. Furthermore, miR-274 is known to be upregulated in Drosophila under pathogen bacteria, Micrococcus luteus infection conditions [28]. However, the biological functions of miR-274 in other contexts and under these upregulated conditions remain unclear. In the present study, we investigated the biological role of Drosophila miR-274 in larval fat bodies in terms of melanotic mass formation and developmental growth. We found that miR-274 overexpression results in the activation of the JNK and JAK/STAT signaling pathways, which are closely associated with the observed phenotypic consequences. Furthermore, we revealed that this regulation of miR-274 was mediated by the RNA-binding protein Fne, which is a biologically relevant target of miR-274. Overall, our findings suggest that miR-274 plays a crucial role in regulating the fne-JNK signaling axis under its upregulated conditions, which in turn affects melanotic mass formation and developmental growth.

Analysis of Melanotic Mass
All larvae and flies were maintained on standard cornmeal/agar media. Melanotic masses were analyzed in wandering third-instar larvae and adult flies as previously described [29]. For quantitative analysis, the percentage of wandering third-instar larvae with melanotic masses was determined using three vials per genotype. Representative images of wandering third-instar larvae or adult flies were captured using a stereomicroscope (Olympus, Shinjuku-ku, Tokyo, Japan).

Determination of the Eclosion Rate
After the eggs were laid on standard cornmeal/agar media at 25 • C, three vials containing eggs from each genotype were transferred to a 29 • C incubator. More than 130 pupae were analyzed for each genotype at 15 d AEL, and the rate of empty puparia in each vial was calculated to determine the eclosion rate.

RNA Isolation and Determination of RNA Transcript Level
Total RNA was purified from S2 cells, larval fat bodies, and adult heads using the TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), according to the manufacturer's instructions. TRI Reagent BD (Molecular Research Center) was used for RNA isolation from larval hemocytes. The following protocol was used for each sample: S2 cells were harvested via centrifugation for 5 min at 350× g. Fat bodies were dissected in cold phosphate-buffered saline (PBS) from wandering third-instar larvae in cold PBS. To collect hemolymph-containing hemocytes, we used a previously described protocol with some  [30]. Briefly, after washing the larvae with distilled water, individual larvae were transferred to cold PBS, and the cuticle was gently torn away to allow the hemolymph to bleed out. Hemolymph was then collected in a new tube. miRNA quantification was performed using a PCR-based method, as previously described [31]. In brief, polyadenine was added to the 3 -end of RNAs using E. coli poly(A) polymerase (Enzynomics, Daejeon, Republic of Korea), and the polyadenylated RNAs were reverse-transcribed using M-MLV Reverse Transcriptase (Enzynomics) and an miR-RTadapter-primer (Supplementary Table S1). To measure the mRNA transcript levels, RNA was first treated with DNase I (Enzynomics) to remove genomic DNA contaminants. RNA was then reverse-transcribed using M-MLV Reverse Transcriptase and random hexamers (Enzynomics). Quantitative PCR was performed using a BioFACT Real-Time PCR Master Mix (BIOFACT, Daejeon, Republic of Korea). To detect the isoforms of fne transcripts, RT-PCR was performed using a MegaFi Fidelity 2× PCR Master Mix (Applied Biological Materials, Richmond, BC, Canada) or BioFACT TM 2× Taq PCR Master Mix (BIOFACT). PCR products were loaded into 2.5% agarose gel and then stained using SYBR TM Safe (Thermo Fisher Scientific, Waltham, MA, USA). The band images were captured on a blue light transilluminator (miniPCR, Cambridge, MA, USA). The primer sequences used for semi-or quantitative-RT-PCR are listed in Supplementary Table S1.

Body Weight and Wing Analysis
Groups of 8-10 adult flies of each genotype (3-5 days old) were transferred to new 1.5 mL tubes and weighed using an analytical balance (Mettler Toledo, Columbus, OH, USA). Four biological replicates per genotype and sex were analyzed.
The left wings of adult female (5 days old) were used for wing analysis. After capturing images using a stereomicroscope (Olympus), the relative wing size was measured using ImageJ software version 1.53 [33]. The wing cell size was analyzed as previously described [34]. The average wing cell size was calculated by dividing the specific wing area by the number of cells in that area. The total cell number of wings was estimated by calculating the wing size and total wing cell number.

Analysis of the Fat Body
To capture an image of the whole larval fat body, the fat body was dissected from five wandering third-instar larvae of each genotype and photographed using a stereomicroscope (Olympus).

Luciferase Reporter Assay
To generate plasmid constructs for the reporter assay, the genomic region of the fne 3 -UTR was amplified by PCR. The amplified DNA fragment was then inserted downstream of the Renilla luciferase in the psiCHECK-2 vector (Promega, Madison, WI, USA). For the mutant form of the fne 3 -UTR, site-directed mutagenesis was performed using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific), as previously described [36]. To generate the miR-274-overexpressing construct, a DNA fragment containing pre-miR-274 was amplified by PCR and cloned into the pMT/V5-His A vector (Invitrogen, Waltham, MA, USA). Both the miRNA-expressing and luciferase reporter constructs were co-transfected using TransIT ® -Insect Transfection Reagent (Mirus Bio, Madison, WI, USA). The activities of Renilla and firefly luciferase were determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) 48 h after miR-274 expression. The Renilla luciferase activity was normalized to the firefly luciferase activity. All primer sets for the reporter assay are listed in Supplementary Table S1.

miR-274 Is Associated with Melanotic Mass Formation through the JNK-JAK/STAT Signaling Pathway Axis
The expression of miR-274 exhibits dynamic changes during Drosophila development [32], and it is also upregulated under pathogen bacteria infection conditions [28]. Thus, we sought to investigate the biological roles of miR-274 under these upregulated conditions. To specially determine the biological roles of miR-274 in the fat bodies of Drosophila, we employed Cg-GAL4 to overexpress miR-274 (hereafter, Cg>miR-274). Cg-GAL4 is known to be active in fat bodies [37], where miR-274 is endogenously expressed (Supplementary Figure S1A). In the fat body of Cg>miR-274 larvae, the expression of miR-274-5p, the major strand of miR-274, was significantly upregulated ( Figure 1A). However, there was no significant increase in the expression level of miR-274-3p compared to the control (Supplementary Figure S1B). Interestingly, ninety-four percent of the Cg>miR-274 larvae exhibited black masses throughout their bodies, whereas the Cg/+ control larvae did not exhibit such melanotic masses ( Figure 1B). The Cg>miR-274 larvae had varying numbers and sizes of melanotic masses throughout their bodies ( Figure 1C). These black masses persisted in the abdomens of both male and female flies ( Figure 1D). In addition, we observed the formation of melanotic masses in the larvae when miR-274 was overexpressed using another fat body-specific GAL4 driver, ppl-GAL4 (Supplementary Figure S1C). Taken together, these results suggest that miR-274 is associated with the formation of melanotic masses.
We proceeded to elucidate the molecular mechanisms underlying melanotic mass formation. According to previous reports, melanotic mass formation is strongly associated with the JAK/STAT signaling pathway [10]. Therefore, we sought to determine whether the overexpression of miR-274 could alter the activity of the JAK/STAT signaling pathway. First, we measured the expression of socs36E mRNA, a target gene of the JAK/STAT signaling pathway [10,38], in the larval fat body of Cg>miR-274. Indeed, socs36E mRNA transcripts were significantly upregulated in the fat body of Cg>miR-274 larvae compared to that in the control larval fat body ( Figure 1E). These results suggest that the overexpression of miR-274 leads to the activation of the JAK/STAT signaling pathway in the fat body. We proceeded to elucidate the molecular mechanisms underlying melanotic mass formation. According to previous reports, melanotic mass formation is strongly associated with the JAK/STAT signaling pathway [10]. Therefore, we sought to determine whether the overexpression of miR-274 could alter the activity of the JAK/STAT signaling pathway. First, we measured the expression of socs36E mRNA, a target gene of the JAK/STAT signaling pathway [10,38], in the larval fat body of Cg>miR-274. Indeed, socs36E mRNA transcripts were significantly upregulated in the fat body of Cg>miR-274 larvae compared to that in the control larval fat body ( Figure 1E). These results suggest that the overexpression of miR-274 leads to the activation of the JAK/STAT signaling pathway in the fat body.
Next, we wondered how miR-274 could affect the JAK/STAT signal pathway. Thus, we determined the mRNA transcript levels of upds, a ligand of the JAK/STAT signaling pathway [39,40], in the larval fat body of Cg>miR-274. Interestingly, we found a significant upregulation of upd3 mRNA transcripts in the fat body of Cg>miR-274 larvae, with an approximately 30.4-fold increase compared to that in the controls ( Figure 1F). In contrast, Next, we wondered how miR-274 could affect the JAK/STAT signal pathway. Thus, we determined the mRNA transcript levels of upds, a ligand of the JAK/STAT signaling pathway [39,40], in the larval fat body of Cg>miR-274. Interestingly, we found a significant upregulation of upd3 mRNA transcripts in the fat body of Cg>miR-274 larvae, with an approximately 30.4-fold increase compared to that in the controls ( Figure 1F). In contrast, the expression of upd2 was downregulated with a relatively smaller change of approximately 4.0-fold in the fat body of Cg>miR-274 larvae compared to that in the controls ( Figure 1G). Furthermore, no expression of upd1 was detected in the fat body. These data indicate that the overexpression of miR-274 leads to a significant upregulation of upd3, which activates the JAK/STAT signaling pathway.
The expression of the upd3 cytokine is induced by the JNK signaling pathway [11]. Thus, to examine whether miR-274 is linked to the activation of the JNK signaling pathway, we determined the level of active phospho-JNK (p-JNK) in the fat body of Cg>miR-274 larvae. The p-JNK level was found to increase in the larval fat body of Cg>miR-274 relative to that in the control fat body ( Figure 1H, Supplementary Figure S2), suggesting that miR-274 overexpression also activates the JNK signaling pathway.
Activation of the JNK signaling pathway in other tissues can upregulate the expression of upd3, which triggers a systemic response in hemocytes. This response can increase hemocyte proliferation and lamellocyte differentiation, ultimately resulting in melanotic mass formation [10,11]. Therefore, to investigate whether the population of lamellocytes, which are closely associated with melanotic mass, is increased in the hemolymph of Cg>miR-274 larvae, we determined the level of Integrin betanu subunit (Itgbn), which is a marker gene of lamellocytes [41] using RT-qPCR. Remarkably, the expression level of Itgbn was significantly higher in the hemocytes of Cg>miR-274 larvae than in the control ( Figure 1I), suggesting that lamellocytes were more differentiated in the hemolymph of Cg>miR-274 larvae. Collectively, our data suggest that miR-274 is associated with melanotic mass formation by regulating the JNK-JAK/STAT pathway axis under its upregulated conditions.

Fat Body Overexpression of miR-274 Leads to Growth Reduction through Defects in the Fat Body
We additionally investigated the phenotypic consequences observed in the fat bodies of Cg>miR-274 larvae. Interestingly, the overall size of the fat bodies expressing miR-274 was markedly reduced compared to that of the control fat bodies ( Figure 2A). Moreover, the size of cells in the fat body was notably reduced in Cg>miR-274 larvae compared to that in the control larvae ( Figure 2B,C). These data indicate that the overexpression of miR-274 causes a reduction in the tissue growth of the larval fat body in addition to melanotic mass formation. Activation of the JAK/STAT signaling pathway is linked to the downregulation of Insulin-like receptor (InR) mRNA expression [42]. Because the JAK/STAT signaling activation was observed in the fat body of Cg>miR-274 larvae, we sought to determine whether the expression of InR mRNA is changed in the fat body by miR-274 overexpression. As expected, the expression of InR mRNA was reduced in the fat body of Cg>miR-274 larvae compared with the controls (Figure 2D), indicating that a reduced size of the fat body by miR-274 overexpression may be likely associated with a decrease in InR mRNA expression.
As the fat body is a crucial tissue associated with energy metabolism and growth [43], we continuously monitored the effects of miR-274 overexpression on developmental growth when miR-274 was overexpressed in the fat body. Most Cg>miR-274 larvae developed into the pupal stage; however, more than 50% of these pupae could not eclose into adult flies ( Figure 2E), indicating that miR-274-induced defects in tissue growth of the larval fat body affect normal development.
Interestingly, the growth of Cg>miR-274 adult flies was reduced relative to that of control flies. To further explore this reduction in growth, we compared the body weights of 3-5-day-old flies between Cg/+ and Cg>miR-274. In both males and females, we observed a significant reduction in the body weight of Cg>miR-274 flies (8.1%-26.5%) compared to Cg/+ control flies ( Figure 2F). Furthermore, in proportion to body weight, the size of Cg>miR-274 adult wings remarkably decreased relative to the size of control wings ( Figure 2G,H). To determine whether the reduction in whole wing size was due to the size and/or number of wing cells, we analyzed the cell size and number of wings in the indicated genotypes. Both the cell size and number of wings of Cg>miR-274 flies were lower than those in the control flies ( Figure 2I,J). Taken together, considering previous results of the upregulation of miR-274 at the larval-to-pupal transitions [32], these results indicate that the untimely upregulation of miR-274 results in the inhibition of growth through the reduction in total tissue mass in the fat body. As the fat body is a crucial tissue associated with energy metabolism and growth [43], we continuously monitored the effects of miR-274 overexpression on developmental growth when miR-274 was overexpressed in the fat body. Most Cg>miR-274 larvae developed into the pupal stage; however, more than 50% of these pupae could not eclose into adult flies ( Figure 2E), indicating that miR-274-induced defects in tissue growth of the larval fat body affect normal development.
Interestingly, the growth of Cg>miR-274 adult flies was reduced relative to that of control flies. To further explore this reduction in growth, we compared the body weights of 3-5-day-old flies between Cg/+ and Cg>miR-274. In both males and females, we observed a significant reduction in the body weight of Cg>miR-274 flies (8.1%-26.5%) compared to Cg/+ control flies ( Figure 2F). Furthermore, in proportion to body weight, the size of Cg>miR-274 adult wings remarkably decreased relative to the size of control wings (Figure 2G,H). To determine whether the reduction in whole wing size was due to the size and/or number of wing cells, we analyzed the cell size and number of wings in the indicated genotypes. Both the cell size and number of wings of Cg>miR-274 flies were lower than those in the control flies ( Figure 2I,J). Taken together, considering previous results of

miR-274 Negatively Regulates the Expression of Fne
We wondered how miR-274 controls melanotic mass formation and growth and thus investigated the regulatory mechanism of miR-274. Using the miRNA target prediction tool, TargetScanFly [9], we first searched for potential target genes that could be regulated by miR-274-5p, which is the main strand of miR-274. We found 173 transcripts with conserved miR-274-5p binding sites (Supplementary Table S2). Among these, based on a screening study of the gene network regulating blood cell homeostasis in Drosophila [2], we selected found in neurons (fne) that encodes an RNA-binding protein associated with the regulation of mRNA processing, such as splicing and alternative polyadenylation (APA) of mRNA [26,44]. According to the prediction using TargetScanFly, miR-274-5p could be bound to two potential sites in the fne 3 -UTR: one conserved site (miR-274-5p BS1) and one poorly conserved site (miR-274-5p BS2) ( Figure 3A, top). served miR-274-5p binding sites (Supplementary Table S2). Among these, based on a screening study of the gene network regulating blood cell homeostasis in Drosophila [2], we selected found in neurons (fne) that encodes an RNA-binding protein associated with the regulation of mRNA processing, such as splicing and alternative polyadenylation (APA) of mRNA [26,44]. According to the prediction using TargetScanFly, miR-274-5p could be bound to two potential sites in the fne 3′-UTR: one conserved site (miR-274-5p BS1) and one poorly conserved site (miR-274-5p BS2) ( Figure 3A, top).  In Drosophila, fne mRNAs are mainly expressed in the nervous system and exist as several isoforms with different lengths of the 3 -UTR [26]. Thus, we determined whether fne mRNA is also expressed in the larval fat body in addition to the nervous system. By semi-RT-qPCR using two different primer sets, we detected fne mRNA with a short and extended 3 -UTR in the fat body of wandering third-instar larvae and adult heads. Consistent with previous results [24,26], the fne mRNA transcript with an extended 3 -UTR was detected in adult heads and was also expressed in the fat body ( Figure 3A, bottom; Supplementary Figure S2). Under Elav-depleted conditions, the nucleus-localized Fne (nFne) bearing the microexon can be induced, which regulates the APA process [26,44]. Therefore, we investigated whether nfne is expressed in the fat body, which is a tissue that does not express Elav. Interestingly, we found that only the nfne transcript containing the microexon was expressed in the larval fat bodies, whereas both general fne and nfne transcripts were expressed at high and low levels, respectively, in adult heads ( Figure 3B, Supplementary Figure S2). These findings suggest that the APA-related nFne likely functions in the larval fat body.
We next examined whether miR-274 negatively regulates the expression of fne in larval fat bodies. Accordingly, we determined the levels of fne mRNA transcripts in the fat bodies of Cg/+ and Cg>miR-274 larvae. As expected, the expression of fne mRNA was significantly reduced in the larval fat bodies overexpressing miR-274 compared to that in the control fat bodies ( Figure 3C). These data support the hypothesis that miR-274 suppresses fne mRNA expression in the larval fat bodies.
To clarify the regulatory interaction between miR-274 and fne mRNA, we performed a luciferase reporter assay. When miR-274 was overexpressed in S2 cells, the activity of Renilla luciferase (RL) fused with wild-type fne 3 -UTR significantly decreased ( Figure 3D,E). In contrast, the inhibitory activity of miR-274 was partially reduced upon co-expression of RL fused with the fne 3 -UTR containing a mutation in the conserved miR-274-5p binding site (miR-274-5p BS1) ( Figure 3E). Taken together, our results suggest that miR-274-5p directly suppresses fne expression in Drosophila.

Fne Is Involved in Melanotic Mass Formation as the Biological Target of miR-274
As fne is suppressed by miR-274 as a direct target in Drosophila, we sought to determine whether the loss of fne leads to phenotypic consequences similar to the defects caused by miR-274 driven by Cg-GAL4. The previous RNAi screening study reported that RNA splicing-associated protein fne may be associated with melanotic mass formation [2]. To test this, we first knocked down fne expression in the fat body using Cg-GAL4 (Cg>fne-RNAi) ( Figure 4A). Consistent with the results for Cg>miR-274 larvae, we observed that melanotic masses remarkably appeared throughout the body of Cg>fne-RNAi larvae ( Figure 4B), and 42.3% of the Cg>fne-RNAi larvae exhibited melanotic mass formation ( Figure 4C). Melanotic masses also persisted in both sexes into adulthood ( Figure 4D). These findings suggest that the loss of fne is associated with melanotic mass formation.   We next examined whether the depletion of fne causes an increase in the activity of the JAK/STAT signaling pathway in the larval fat body, similar to Cg>miR-274 larvae. The expression level of socs36E was significantly higher in the fat bodies of Cg>fne-RNAi larvae than in the control fat bodies ( Figure 4E), indicating that the depletion of fne activates the JAK/STAT signaling pathway. Furthermore, the expression of upd3 was markedly upregulated in the fat bodies of Cg>fne-RNAi larvae with an approximately 27.2-fold increase compared to that in the controls ( Figure 4F). In contrast, the expression of upd2 was downregulated, with an approximately 2.9-fold decrease compared to that in the controls ( Figure 4G). These results suggest that fne is involved in the activation of the JAK/STAT signaling pathway through a significant upregulation of upd3 expression rather than upd2 expression.
Furthermore, we determined whether fne alters the level of active p-JNK, as observed with miR-274 overexpression. The level of active p-JNK increased in the fat body of Cg> fne-RNAi larvae compared to that in the control ( Figure 4H, Supplementary Figure S2), indicating that fne depletion activates the JNK signaling pathway.
We investigated whether the reduction in fne expression leads to increased lamellocyte differentiation. We determined the levels of Itgbn mRNA in the hemolymph of Cg>fne-RNAi larvae. Consistent with Cg>miR-274 larvae, Itgbn mRNA levels were higher in the hemocytes of Cg>fne-RNAi larvae than in that of the controls ( Figure 4I). Thus, the results imply that the depletion of fne causes an increase in the number of lamellocytes. Collectively, our data suggest that fne, as a biological target of miR-274, is associated with melanotic mass formation through the JNK-JAK/STAT signaling pathway.

Fne Depletion Leads to Growth Reduction
We investigated whether fne is also implicated in developmental growth, similar to miR-274. First, we analyzed the total fat body mass in Cg>fne-RNAi larvae. Similar to Cg>miR-274 larvae, the overall size of the fat body was remarkably reduced in Cg>fne-RNAi larvae compared to that in control larvae ( Figure 5A). To further examine this phenotype, we compared the sizes of fat body cells between Cg/+ and Cg>fne-RNAi larvae after Factin staining with phalloidin. The cell size of the fat body in Cg>fne-RNAi larvae was significantly smaller than that in the controls ( Figure 5B,C). In addition, consistent with the observations from the fat body of Cg>miR-274 larvae, the expression of InR mRNA was significantly reduced in the fat body of Cg>fne-RNAi larvae compared with the controls ( Figure 5D). These results suggest that the reduced fat body resulting from fne depletion may be likely associated with a decrease in InR mRNA expression. notype, we compared the sizes of fat body cells between Cg/+ and Cg>fne-RNAi larvae after F-actin staining with phalloidin. The cell size of the fat body in Cg>fne-RNAi larvae was significantly smaller than that in the controls ( Figure 5B,C). In addition, consistent with the observations from the fat body of Cg>miR-274 larvae, the expression of InR mRNA was significantly reduced in the fat body of Cg>fne-RNAi larvae compared with the controls (Figure 5D). These results suggest that the reduced fat body resulting from fne depletion may be likely associated with a decrease in InR mRNA expression. The eclosion rate of Cg>fne-RNAi pupae was remarkably reduced compared to that of control pupae ( Figure 5E). To investigate whether defects in the larval fat body of Cg>fne-RNAi affected developmental growth, we measured the body weights of Cg>fne-RNAi flies. Consistent with the reduction in body weight in Cg>miR-274 flies, Cg>fne-RNAi flies of both sexes displayed significantly lower body weight than control flies ( Figure 5F). Moreover, we observed a decrease in the whole size of Cg>fne-RNAi wings relative to control wings in proportion to body weight ( Figure 5G,H). The size and the total number of wing cells in Cg>fne-RNAi flies were reduced compared to those in control flies ( Figure 5I,J). Together, these observations indicate that fne, a target of miR-274, also plays a role in developmental growth.

Discussion
In this study, we demonstrated that miR-274 activates the JNK-JAK/STAT signaling axis by targeting fne, which in turn controls the formation of melanotic mass. However, further studies are needed to address the detailed mechanism by which fne controls the JNK-JAK/STAT signaling pathways. Based on previous reports [26], Fne may regulate gene expression through its involvement as an RNA-binding protein in the APA process. In particular, the nucleus-localized nFne is involved in this process under Elav-depleted conditions [26,44]. Based on our results, the nFne isoform is likely functional in the APA process in the larval fat body, where Elav is not expressed. Thus, the depletion of fne may induce a shortened 3 -UTR of fne-target mRNAs, thereby reducing the chance of negative regulation by miRNAs; this is because miRNAs mainly suppress the expression of target genes by binding to the 3 -UTR of mRNAs [14], which may result in an increase in the expression of genes targeted by Fne. By the targets of RNA-binding proteins identified by editing (TRIBE) method [45], target mRNAs directly bound by Fne have been identified in Drosophila S2 cells [46]. The identified genes include Stat92E, a key factor in the JAK/STAT signaling pathway, and positive regulators of JNK signaling, such as Cell division cycle 42 (Cdc42), Rac1, and cryptocephal (crc). Thus, changes in the length of the 3 -UTR of these genes involved in the JNK or the JAK/STAT signaling pathway, which are regulated by Fne, may alter their expression level.
Our findings indicate that miR-274 activates JNK signaling by suppressing fne, which in turn induces the expression of upd3 in the fat body. Subsequently, Upd3 derived from the fat body may stimulate lamellocyte differentiation, triggering melanotic mass formation. Although we provided evidence of the association between miR-274 overexpression in the fat body and melanotic mass formation based on observations using Cgand ppl-GAL4 drivers, we cannot completely exclude the possibility that miR-274 may regulate the JNK-JAK/STAT signaling pathway in blood cells in addition to the fat body. The Cg-GAL4 driver primarily activates the fat body but also drives gene expression in blood cells. [37]. Furthermore, the effects of miR-274 on melanotic mass formation were relatively weaker in ppl>miR-274 larvae compared to Cg>miR-274 larvae. Thus, interactions between the regulatory networks of the fat body and hemocytes may synergistically influence melanotic mass formation.
We noted a significant reduction in tissue size and activation of the JNK-JAK/STAT signaling pathway in the fat body when miR-274 and fne were overexpressed and depleted, respectively. In addition to JAK/STAT signaling, JNK signaling is well known to play a pro-apoptotic role in cell death and negatively regulates insulin/IGF-like signaling in both mammals and Drosophila [47,48]. The activation of JNK signaling has been demonstrated to cause defects in the normal growth and development of the wing and eye in Drosophila [49,50]. These findings provide support for the association between miR-274, fne, and developmental growth. In addition, a previous report described that knockdown of upd2 in the fat body, but not upd1 and upd3, leads to growth reduction by regulating the secretion of Drosophila insulin-like peptides in the insulin-producing cells [51]. Therefore, as observed in this study, the downregulation of upd2 in the fat body may affect growth reduction.
According to previous reports, the expression of pre-miR-274 is higher in the glia than in other tissues during the larval stage of Drosophila [27]. However, mature-miR-274 is released as an exosome and broadly distributed to other cells, including synaptic boutons, muscle cells, and tracheal cells [27]. This finding implies that glia-derived exosomal mature-miR-274 could circulate in the larval hemolymph and affect the expression of its target genes in several target tissues, such as hemocytes and fat bodies, along with miR-274 transcribed in the tissue itself. The biological target gene of miR-274, fne, is primarily expressed in neuronal tissue in Drosophila [24]. The expression of both miR-274 and fne in the same tissues indicates that they are likely to maintain a regulatory network, which is similar to our findings in fat bodies. Based on our previous results, the expression of miR-274 is upregulated during the larval-to-pupal transition [32], which suggests that changes in miR-274 expression may alter the composition of the extended 3 -UTR of neuronal-specific genes by regulating the expression of fne during this developmental stage. Thus, future studies should investigate the perturbation of APA in neuron-specific genes during metamorphosis.
We observed a reduction in both fne mRNA and reporter activity with the fne 3 -UTR when miR-274 was overexpressed. miR-274-5p was found to negatively regulate the expression of fne at the transcript level by binding to at least one conserved site (miR-274-5p BS1) in the 3 -UTR of fne mRNA. However, the luciferase reporter activity suppressed by miR-274 was found to be partially restored when the region corresponding to the miR-274-5p seed was mutated in the miR-274-5p BS1. This result implies that other binding sites may exist for miR-274-5p in the fne 3 -UTR. The poorly conserved binding site (miR-274-5p BS2) in the fne 3 -UTR could be a potential regulatory binding site for miR-274-5p. Future studies could examine whether miR-274-5p targets miR-274-5p BS2 in the fne 3 -UTR.
A relatively stronger formation of melanotic masses was observed in larvae overexpressing miR-274 than in fne-depleted larvae. Most Cg>miR-274 larvae exhibited a melanotic mass, whereas less than 50% of Cg>fne-RNAi larvae had black dots. The melanotic masses in the individual Cg>miR-274 larvae were numerous and larger than those in the Cg>fne-RNAi larvae. Such small differences between the lines linked in the regulatory network may arise from other genes affected by alterations in miR-274 or fne expression. TargetScanFly identified 173 potential miR-274-5p targets. Some of these targets, such as S-adenosylmethionine Synthetase (Sam-S) and slimfast (slif ), might directly or indirectly affect the JNK and/or JAK/STAT signaling pathways. In addition, although miR-274-3p is not the predominant strand of miR-274, miR-274-3p might contribute to melanotic mass formation and/or growth. TargetScanFly identified numerous potential target genes of miR-274-3p. For example, GO term analysis using DAVID [52,53] revealed 11 genes (Connector of kinase to AP-1, Menin 1, Protein phosphatase V, Ras-like protein A, Striatin interacting protein, alphabet, capping protein alpha, fiery mountain, flapwing, icarus, and pebbled) that can play negative regulators of the JNK signaling pathway.
Collectively, our findings provide insights into the potential molecular mechanisms underlying these phenomena. Further studies should explore these mechanisms in detail to provide a more comprehensive understanding of the physiological processes.

Conclusions
In this study, under its overexpression conditions, Drosophila miR-274 was found to play a regulatory role in the JNK and JAK/STAT signaling pathways by suppressing the expression of fne encoding an RNA-binding protein that functions in RNA processing, such as APA and alternative splicing. This regulatory network of miR-274-fne-JNK signaling in the larval fat body is associated with the formation of melanotic mass and developmental growth. Thus, our findings provide valuable insights into the molecular mechanisms underlying melanotic mass formation and growth. Furthermore, a better understanding of this regulatory mechanism will contribute to the development of novel ideas for regulating immune defense responses against invading pathogens and growth regulation.  Figure S1: Expression of miR-274, and the formation of melanotic masses in ppl>miR-274 larvae. Supplementary Figure S2: Images of Western blotting and DNA gel presented in the main figures.