Abstract:Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone‐responsive miRNA, miR‐252, is normally upregulated during the pupal and adult stages of Drosophila development. Here, we found that overexpression of miR‐252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decr… Show more
“…Pxy-miR-252 belonged to the miR-252 family. It has been found that miR-252 could directly repress the mbt expression to control the developmental growth of Drosophila ( Lim et al, 2019 ) and was also involved in dengue virus replication in Aedes albopictus ( Yan et al, 2014 ). Pxy-miR-274 was classified into the miR-274 family.…”
The diamondback moth (DBM), Plutella xylostella, one of the most destructive lepidopteran pests worldwide, has developed field resistance to Bacillus thuringiensis (Bt) Cry toxins. Although miRNAs have been reported to be involved in insect resistance to multiple insecticides, our understanding of their roles in mediating Bt resistance is limited. In this study, we constructed small RNA libraries from midguts of the Cry1Ac-resistant (Cry1S1000) strain and the Cry1Ac-susceptible strain (G88) using a high-throughput sequencing analysis. A total of 437 (76 known and 361 novel miRNAs) were identified, among which 178 miRNAs were classified into 91 miRNA families. Transcripts per million analysis revealed 12 differentially expressed miRNAs between the Cry1S1000 and G88 strains. Specifically, nine miRNAs were down-regulated and three up-regulated in the Cry1S1000 strain compared to the G88 strain. Next, we predicted the potential target genes of these differentially expressed miRNAs and carried out GO and KEGG pathway analyses. We found that the cellular process, metabolism process, membrane and the catalytic activity were the most enriched GO terms and the Hippo, MAPK signaling pathway might be involved in Bt resistance of DBM. In addition, the expression patterns of these miRNAs and their target genes were determined by RT-qPCR, showing that partial miRNAs negatively while others positively correlate with their corresponding target genes. Subsequently, novel-miR-240, one of the differentially expressed miRNAs with inverse correlation with its target genes, was confirmed to interact with Px017590 and Px007885 using dual luciferase reporter assays. Our study highlights the characteristics of differentially expressed miRNAs in midguts of the Cry1S1000 and G88 strains, paving the way for further investigation of miRNA roles in mediating Bt resistance.
“…Pxy-miR-252 belonged to the miR-252 family. It has been found that miR-252 could directly repress the mbt expression to control the developmental growth of Drosophila ( Lim et al, 2019 ) and was also involved in dengue virus replication in Aedes albopictus ( Yan et al, 2014 ). Pxy-miR-274 was classified into the miR-274 family.…”
The diamondback moth (DBM), Plutella xylostella, one of the most destructive lepidopteran pests worldwide, has developed field resistance to Bacillus thuringiensis (Bt) Cry toxins. Although miRNAs have been reported to be involved in insect resistance to multiple insecticides, our understanding of their roles in mediating Bt resistance is limited. In this study, we constructed small RNA libraries from midguts of the Cry1Ac-resistant (Cry1S1000) strain and the Cry1Ac-susceptible strain (G88) using a high-throughput sequencing analysis. A total of 437 (76 known and 361 novel miRNAs) were identified, among which 178 miRNAs were classified into 91 miRNA families. Transcripts per million analysis revealed 12 differentially expressed miRNAs between the Cry1S1000 and G88 strains. Specifically, nine miRNAs were down-regulated and three up-regulated in the Cry1S1000 strain compared to the G88 strain. Next, we predicted the potential target genes of these differentially expressed miRNAs and carried out GO and KEGG pathway analyses. We found that the cellular process, metabolism process, membrane and the catalytic activity were the most enriched GO terms and the Hippo, MAPK signaling pathway might be involved in Bt resistance of DBM. In addition, the expression patterns of these miRNAs and their target genes were determined by RT-qPCR, showing that partial miRNAs negatively while others positively correlate with their corresponding target genes. Subsequently, novel-miR-240, one of the differentially expressed miRNAs with inverse correlation with its target genes, was confirmed to interact with Px017590 and Px007885 using dual luciferase reporter assays. Our study highlights the characteristics of differentially expressed miRNAs in midguts of the Cry1S1000 and G88 strains, paving the way for further investigation of miRNA roles in mediating Bt resistance.
“…To test the efficiency of RNAi lines for Vha knockdown, flies carrying either hs‐GAL4 only ( hs‐GAL4 /+) or both hs‐GAL4 and each of the experimental UAS‐RNAi transgenes ( hs > Vha‐RNAi ) were heat‐shocked at the wandering L3 stage at 37°C for 40 minutes and then cultured at 25°C for 36 hours prior to analysis. A flip‐out clonal assay was performed by crossing hsFLP; Actin > CD2 > GAL4 , UAS‐nlsGFP flies with the experimental UAS transgenic flies or w 1118 flies, as described previously 25 …”
Section: Methodsmentioning
confidence: 99%
“…Target sites for miRNA binding in the 3′‐UTR of the corresponding genes were assessed by a luciferase reporter assay as described previously 25 . Primer sets that were used to construct the luciferase reporters are listed in Table S4.…”
Section: Methodsmentioning
confidence: 99%
“…After puparium formation, defective anterior spiracle eversion was examined as described previously, 25 and the percentage of pupal death was determined by counting the empty pupal cases and dead flies after living adult flies were emptied from the vials.…”
Section: Methodsmentioning
confidence: 99%
“…For example, the expression of let‐7‐Complex miRNAs is induced by ecdysone signaling, and these miRNAs target abrupt and chinmo to control the distinct metamorphic processes 22-24 . In another ecdysone‐responsive miRNA, miR‐252, expression is upregulated during the pupal and adult stages of development, and it targets mbt to control the developmental growth 25 . However, a comprehensive understanding of metamorphosis‐associated miRNAs and their biologically relevant targets remains elusive.…”
The steroid hormone ecdysone is the central regulator of insect metamorphosis, during which a growing, immature larva is remodeled, through pupal stages, to a reproductive adult. However, the underlying mechanisms of ecdysone-mediated metamorphosis remain to be fully elucidated. Here, we identified metamorphosis-associated microRNAs (miR-NAs) and their potential targets by cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 protein in Drosophila. Interestingly, miR-8-3p targeted five Vha genes encoding distinct subunits of vacuolar H + -ATPase (V-ATPase), which has a vital role in the organellar acidification. The expression of ecdysone-responsive miR-8-3p is normally downregulated during Drosophila metamorphosis, but temporary overexpression of miR-8-3p in the whole body at the end of larval development led to defects in metamorphosis and survival, hallmarks of aberrant ecdysone signaling.In addition, miR-8-3p was expressed in the prothoracic gland (PG), which produces and releases ecdysone in response to prothoracicotropic hormone (PTTH). Notably, overexpression of miR-8-3p or knockdown of its Vha targets in the PG resulted in larger than normal, ecdysone-deficient larvae that failed to develop into the pupal stage but could be rescued by ecdysone feeding. Moreover, these animals showed defective PTTH signaling with a concomitant decrease in the expression of ecdysone biosynthetic genes. We also demonstrated that the regulatory network between the conserved miR-8-3p/miR-200 family and V-ATPase was functional in human cells. Consequently, our data indicate that the coordinated regulation of V-ATPase subunits by miR-8-3p is involved in Drosophila metamorphosis by controlling the ecdysone biosynthesis. K E Y W O R D S Ago1, CLIP-seq, ecdysone, metamorphosis, microRNA, V-ATPase 6450 | LIM et aL.
BACKGROUDGaleruca daurica has become a new pest on the Inner Mongolia grasslands since its abrupt outbreak in 2009, and caused serious damage. As a pupa indicator during insect metamorphosis, the early response gene of ecdysone signaling pathway, Broad‐Complex (Br‐C), plays a vital role in the growth and development of insects. MicroRNAs (miRNA) are small non‐coding RNAs which mediate various biological activities. However, it is unknown whether and how Br‐C is regulated by miRNAs.RESULTSTemporal expression profiles revealed that miR‐285 and Br‐C basically displayed an opposite trend during the larval‐adult development, and Br‐C was sharply up‐regulated on the last day of final instar larvae meanwhile miR‐285 was significantly down‐regulated. Both dual‐luciferase reporter assay and miRNA‐mRNA interaction assay indicated that miR‐285 interacts with the coding sequence of Br‐C and represses its expression. Not only overexpression but also downexpression of miR‐285 led to the failure of larval to pupal to adult metamorphosis. In addition, both overexpression of miR‐285 and silence of Br‐C inhibited the expression of Br‐C and other ecdysone signaling pathway genes including E74, E75, ECR, FTZ‐F1, and HR3. On the contrary, suppressing miR‐285 obtained opposite results. Further experiments showed that 20‐hydroxyecdysone down‐regulated miR‐285, and up‐regulated Br‐C and above‐mentioned genes whereas JHA resulted in opposite effects.CONCLUSIONOur results reveal that miR‐285 is involved in mediating the metamorphosis in G. daurica by targeting Br‐C in the ecdysone signaling pathway. miR‐285 and its target Br‐C could be as a potential target for G. daurica management.
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