MiR-11 Regulates Pupal Size Of Drosophila Melanogaster Via Directly Targeting Ras85D

Metamorphosis is the species-typical and most important phenomenon of insects,which is caused by a long-term adaptive evolution of insects to the external environment. According to the presence of pupal stage in the development, insects are divided into two categories: incomplete metamorphosis and complete metamorphosis.Complete metamorphosis insects locate on the highest degree in evolution, which have four stages: egg, larva, pupa and adult. Particularly, adult morphology and size are determined by the individual pupae size. However, which factors affect the size of the pupae? Previous studies showed that the pupa length and body size involve in a series of gene expression, network regulation and hormone releasing, which is an extremely complex process. So far, the understand to the mechanism under this process could be the tip of the iceberg, especially which miRNAs could regulate pupal development is poorly understood. MicroRNAs (miRNAs) are a class of small non-coding RNAs(about 21-24 nucleotides) which play key roles in negatively regulation of gene expression through targeting genes transcripts. It is hard to clear study multiple function of individual miRNA at present due to the numerous target genes and other characteristics of short mature miRNA. So we study miRNA function involved in determination of pupal size via combining bioinformatics with molecular experiment.It is profound theoretical significance for revealing miRNA regulation mechanism in the insects and provide new way to control insect pests in agriculture.Drosophila melanogaster is a complete metamorphosis insects and a classic animal model, which was selected as the object of our study. Drosophila miRNA expression pattern was analyzed, and miR-11 expression in the late third instar larvae was found is higher than the expression in other stages. Further analysis of miR-11 family evolutionary shows that miR-11 is conserved in complete metamorphosis insects, rather than in incomplete metamorphosis insects. These results implied that miR-11 may be involved in the pupal developmental processes in Drosophila melanogaster. To test this hypothesis, following studies were done:1. miR-11 knockout (KO) flies was constructed using RMCE technology and miR-11 loss function or overexpression flies was generated using UAS/Gal4 system. We observed phenotype of miR-11 mutants and found that knockout or loss function of miR-11 could cause increased pupae length and weight, however miR-11 overexpression flies appeared lethal in pupal stage. Through length measurement and phenotypic observation on first instar larvae, second instar larvae, third instar larvae,pupae and adults, developmental delay leading to longer pupae was excluded. miR-11KO mutant showing increased pupal length and adult body size was found, while there is no significant difference on the larval size of miR-11 knockout and wild-type.Knockout or activity inhibition of miR-11 inducing large body size of Drosophila during metamorphosis suggests that miR-11 could involved in regulating the size of pupal length.2. To reveal the mechanisms of miR-11 in regulation pupae length, 189 target genes of miR-11 were performed by pathway enrichment analysis. The analysis showed that these target genes were strong enriched in Ras/MAPK signaling pathway, in which Ras85D and Sos gene could be targeted by miR-11. Further studies on the relation between miR-11 and Ras85D, Sos demonstrated that miR-11 suppresses Ras85D expression via targeting Ras85D 3'UTR in vivo and in vitro, but not acting 3'UTR of Sos gene. The results were speculated that miR-11 may regulate the length of the pupa size through targeting Ras85D.3. In order to testify whether miR-11 directly targeting Ras85D induced the deficient phenotype of the pupal length of Drosophila, further studies about the role of miR-11-Ras85D relation in pupal development were carried out. We constructed double mutations flies, co-inhibition and miR-11 and Ras85D function flies, using basic genetic methods, and found the double mutations flies could rescue deficient phenotype of miR-11KO flies. miR-11 and Ras85D temporal expression pattern analysis showed the expressions between miR-11 and Ras85D have counter-balance relationship. We suggest that inhibition of miR-11 to Ras85D throughout metamorphosis during development, and miR-11 regulates pupal length by directly targeting Ras85D.In summary, we use an effective mean of combining bioinformatics methods and molecular biological technology to prove that miR-11 regulates the pupal length via directly targeting the 3'UTR of Ras85D and therefore participates into the determination of final pupal size in Drosophila metamorphosis processes. The conclusion provides strong and concrete experimental evidence to miRNA regulation of insect metamorphosis, and provides a new angle of view for understanding the regulation mechanisms of metamorphosis. Meanwhile, effectively mean of combining bioinformatics and molecular biology create a new strategy for researching a large number of potentially unknown miRNA function. Further, it is profound and meaningful for agricultural pest control.