Localized expression pattern of miR-184 in Drosophila
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- Li, P., Peng, J., Hu, J. et al. Mol Biol Rep (2011) 38: 355. doi:10.1007/s11033-010-0115-1
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MicroRNAs (miRNAs) are a kind of endogenous non-coding small RNAs whose specific functions in animals are generally important. Although functions of some miRNAs have been identified, the role of miR-184 remains unknown. Here, we determined the temporal and spatial expression pattern of miR-184 during the different development stages and tissues in Drosophila. Strikingly, miR-184 is expressed ubiquitously in Drosophila embryos, larvae and adults, its expression pattern shows a dynamic changes during the development of embryo, especially in the central nervous system. This expression profile suggests that miR-184 may act important function in Drosophila development.
MicroRNAs (miRNAs) are 21–24 nt non-coding RNAs that form duplexes with target mRNA transcripts and induce transcript cleavage and/or inhibit productive translation. Since the identified of the first miRNAs lin-4 in Caenorhabditis elegans in 1993 , it has been reported about 175 miRNAs in worms, 157 miRNAs in flies, 579 miRNAs in mouse and approximately 721 miRNAs in human (http://www.mirbase.org). miRNAs are universally present in animals, plants and viruses that play an important role in the development, proliferation, differentiation, apoptosis of organisms and cancers [2–5].
Most miRNAs display a tissue- and/or developmental-specific expression pattern, which suggests highly specific and diverse roles for miRNAs. For example, miR-1 is found exclusively in muscles, where it regulates cardiac differentiation in Drosophila , miR-9a is specifically expressed in epithelial cells and proved to controls the generation of sensory organs , miR-278 is specifically expressed in adipose cells, where it regulates genes involved in insulin secretion .
miR-184 is a single copy gene and evolutionarily conserved at the nucleotide level from flies to humans. It is expressed in the mesoderm in situ hybridization in Drosophila . In Zebrafish, it is expressed in lens, hatching gland and epidermis by Northern blots using LNA (locked-nucleic acid)-modified DNA oligonucleotide probes . An analysis of the primary transcript of miR-184 (pri-mir-184) in several mouse tissues revealed specific expression in brain and testis, its expression that repressed by the binding of MeCP2 to its promoter, is upregulated by the release of MeCP2 after depolarization, this give a clue to link between the miRNAs and DNA methylation pathways . Over expression of miR-184 in neuroblastoma cell lines results in massive apoptosis . A recent paper discovers miR-184 has multiple roles in Drosophila female germline development . Together, these findings indicate that miR-184 can play essential roles in development. However, the temporal and spatial expression pattern of miR-184 is still being debated.
In this paper, we examined the expression pattern of miR-184 throughout the life cycle and different discs of Drosophila by in situ hybridization and a modified ribonuclease protection assay (RPA) method. We give some clues to the miR-184 studies.
Materials and methods
RNA isolation and reverse transcription (RT-PCR) of whole samples were performed according to standard protocols. An oligo dT18 primer was used for the RT step and the gene-specific primer pairs were used for the PCR. Primer sequences are 5′-TCGGATACGGATACGGATTCCTG-3′ and 5′-ATGCACATGTTGGCAGACAGC-3′. Ten percent of the RT product served as the template for the PCR, which was kept in the linear range of amplification. The products were separated on 2% agrose gel.
In situ hybridization
A 500 bp DNA fragment of miR-184 precursor were amplified from Drosophila genomic DNA by PCR, Primers are 5′-TGCGCACGTTCAATTTGCAA-3′ and 5′-ATGAGTTGGCAGACAGCAGC-3′. The 500 bp fragment was labeled with digoxigenin-UTP (Roche Company) by using the T7 transcriptase in vitro according to the manufacturer’s instructions and used as probe.
Embryos were collected up to 17 h, fixed and prepared by using standard protocols. Embryos were prehybridized for 1 h and then hybridized in 0.2 ng/μl Dig-probe for 12–18 h at 55°C and incubated with anti-Dig Fab alkaline phosphatase coupled antibody overnight at 4°C at a dilution 1:2000. The pri-mir-184 was detected by AP substrate solution (NBT & BCIP).
Ribonuclease protection assay
The ribonuclease protection assay was carried out mainly according to the mirVana miRNA Detection Kit Instructions (Ambion Company) with the following modifications. The miR-184 was detected with a purified biotin-labeled oligo probe by using the T7 RNA polymerase in vitro according to the manufacturer’s instructions, the 5S rRNA was used as control. The probe sequence is 5′-GAGACAGGGCCCUU-AUCAGUUCUCCGUCCA-3′, which contains a 22 nt sequence (GCCCUUAUCA-GUUCUCCGUCCA) that is the complement of the mature miR-184 sequence and 8 nt (GAGACAGG) nonspecific sequence that will not hybridize with the target RNA. Hybridization was performed at 42°C. Un-hybridized probe and RNAs were digested by RNase A/RNase T1. The RNases activity was inactivated by RNase Inactivation/PPT solution and protected RNA fragments were precipitated using ethanol. RNAs were separated on 15% denaturing polyacrylamide gel, transferred to Hybond nylon membrane (Biodyne B) and united with nylon membrane by 1200J UV crossing. miR-184 was probed with Streptividin-HRP (Genscript Company) at a dilution 1:20000. Cross reacting bands were visualized using the ECL detection kit (Thermo Company). The sequence in the probe that does not hybridize to the target is longer than the protected fragment so that an obvious difference in size is seen between the full-length undigested probe and the protected fragment after RNase digestion. The shift in size from full-length to a smaller protected fragment helps to validate that RNA from the sample is protecting the probe and is not an artifact.
Results and discussion
In conclusion, we here describe the temporal and spatial expression pattern of miR-184 in Drosophila development, miR-184 expression in the embryo is neurospecific whereas in the larvae, transcripts are also found in several discs (e.g. head, eye and wing discs). This expression profile suggests that miR-184 may play crucial roles in Drosophila development, including tissue fate establishment, differentiation or the maintenance of tissue identity.
We thank the Bloomington stocks center for fly stocks, this work was supported by NSFC (30670444).