Project description:miRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally. We used small RNA sequencing to identify tissue-specific miRNAs in the adult brain, thorax, gut and fat body of Drosophila melanogaster. One of the most brain-specific miRNAs that we identified was miR-210, an evolutionarily highly conserved miRNA implicated in the regulation of hypoxia in mammals. In Drosophila, we show that miR-210 is specifically expressed in sensory organs including photoreceptors. miR-210 knock-out mutants are not sensitive towards hypoxia but show progressive degradation of photoreceptor cells, accompanied by decreased photoreceptor potential, demonstrating an important function of miR-210 in photoreceptor maintenance and survival.
Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. miR-210 can be considered a master miRNA of hypoxic response and is currently regarded as a promising novel non-invasive tumor hypoxia marker. The targets identified to date indicate that miR-210 plays a role in cell cycle regulation, differentiation, mitochondrial metabolism repression, DNA repair and apoptosis. In order to identify miRNAs sub-sequentely modulated by miR-210, miRNA expression profiles of human umbilical vein endothelial cells (HUVEC) over-expressing miR-210 were generated, allowing the identification of miRNAs modulated upon miR-210 up-regulation. HUVEC over-expressing pre-miR-210 or a scramble sequence were generated by retroviral infection, yielding a selected population that expressed mature miR-210 levels comparable with those observed in hypoxic cells. miRNA expression profiles were then measured and miRNAs modulated upon miR-210 up-regulation were identified. This Sample represents four hybridizations - one of which was a dye-swap.
Project description:MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally. Here, we show that miR-210 is induced by Oct-2, a key transcriptional mediator of B-cell activation. Germline deletion of miR-210 results in the development of autoantibodies from 5 months of age. Overexpression of miR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice over-expressing miR-210 exhibited impaired class-switched antibody responses, a finding confirmed in wild-type B-cells transfected with a miR-210 mimic. In vitro studies demonstrated a defect in cellular proliferation and cell-cycle entry, which was consistent with the transcriptomic analysis demonstrating down-regulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of miR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.
Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3âUTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia. Experiment Overall Design: Gene expression modifications induced by both miR-210 over-expression and blockade were evaluated. In order to identify new direct and indirect miR-210 targets, transcripts repressed by miR-210 over-expression and up-regulated by miR-210 inhibition (and vice versa) were selected.
Project description:A method for the long-term maintenance of germ-free flies was established using aseptic isolators. The methodology effectively and reliably yields large numbers of germ-free flies in homogeneous cultures. Germ-free flies exhibited increased lifespan (only female flies) and decreased egg production, markedly reduced fat storage, less midday sleep, and enhanced aggressiveness (male flies). Fructilactobacillus—a species of fly intestinal microbes—was re-colonized in germ-free flies, and these gnotobiotic flies were successfully maintained for numerous generations. The proteome of those flies were analyzed.