microRNA target predictions across seven Drosophila species and comparison to mammalian targets

microRNAs are small noncoding genes that regulate the protein production of genes by binding to partially complementary sites in the mRNAs of targeted genes. Here, using our algorithm PicTar, we exploit cross-species comparisons to predict, on average, 54 targeted genes per microRNA above noise in Drosophila melanogaster. Analysis of the functional annotation of target genes furthermore suggests specific biological functions for many microRNAs. We also predict combinatorial targets for clustered microRNAs and find that some clustered microRNAs are likely to coordinately regulate target genes. Furthermore, we compare microRNA regulation between insects and vertebrates. We find that the widespread extent of gene regulation by microRNAs is comparable between flies and mammals but that certain microRNAs may function in clade-specific modes of gene regulation. One of these microRNAs (miR-210) is predicted to contribute to the regulation of fly oogenesis. We also list specific regulatory relationships that appear to be conserved between flies and mammals. Our findings provide the most extensive microRNA target predictions in Drosophila to date, suggest specific functional roles for most microRNAs, indicate the existence of coordinate gene regulation executed by clustered microRNAs, and shed light on the evolution of microRNA function across large evolutionary distances. All predictions are freely accessible at our searchable Web site http://pictar.bio.nyu.edu.     

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3'-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5'-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5'-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5'-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3' region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122-HCV interaction, and the broader consequences of miRNAs binding to 5'-UTR sites.     

MicroRNA Regulation of the Synaptic Plasticity-Related Gene Arc

Expression of activity-regulated cytoskeleton associated protein (Arc) is crucial for diverse types of experience-dependent synaptic plasticity and long-term memory in mammals. However, the mechanisms governing Arc-specific translation are little understood. Here, we asked whether Arc translation is regulated by microRNAs. Bioinformatic analysis predicted numerous candidate miRNA binding sites within the Arc 3'-untranslated region (UTR). Transfection of the corresponding microRNAs in human embryonic kidney cells inhibited expression of an Arc 3'UTR luciferase reporter from between 10 to 70% across 16 microRNAs tested. Point mutation and deletion of the microRNA-binding seed-region for miR-34a, miR-326, and miR-19a partially or fully rescued reporter expression. In addition, expression of specific microRNA pairs synergistically modulated Arc reporter expression. In primary rat hippocampal neuronal cultures, ectopic expression of miR-34a, miR-193a, or miR-326, downregulated endogenous Arc protein expression in response to BDNF treatment. Conversely, treatment of neurons with cell-penetrating, peptide nucleic acid (PNA) inhibitors of miR-326 enhanced Arc mRNA expression. BDNF dramatically upregulated neuronal expression of Arc mRNA and miR-132, a known BDNF-induced miRNA, without affecting expression of Arc-targeting miRNAs. Developmentally, miR-132 was upregulated at day 10 in vitro whereas Arc-targeting miRNAs were downregulated. In the adult brain, LTP induction in the dentate gyrus triggered massive upregulation of Arc and upregulation of miR-132 without affecting levels of mature Arc-targeting miRNAs. Turning to examine miRNA localization, qPCR analysis of dentate gyrus synaptoneurosome and total lysates fractions demonstrated synaptic enrichment relative to small nucleolar RNA. In conclusion, we find that Arc is regulated by multiple miRNAs and modulated by specific miRNA pairs in vitro. Furthermore, we show that, in contrast to miR-132, steady state levels of Arc-targeting miRNAs do not change in response to activity-dependent expression of Arc in hippocampal neurons in vitro or during LTP in vivo.     

FOXO1 3′UTR functions as a ceRNA in repressing the metastases of breast cancer cells via regulating miRNA activity

The competitive endogenous RNAs (ceRNAs) are RNA molecules that affect each other’s expression through competition for their shared microRNAs (miRNAs). In this study we explored whether FOXO1 3′UTR can function as a ceRNA in repressing epithelial-to-mesenchymal transition (EMT) and metastasis of breast cancer cells via regulating miR-9 activity. We found that miR-9 binds to both the FOXO1- and E-cadherin-3′UTR, indicating that the FOXO1- and E-cadherin-3′UTR can be linked through miR-9. Follow-up analyses showed that there existed a competition of miR-9 between FOXO1 and E-cadherin-3′UTR. Thus FOXO1 3′UTR inhibits the metastases of breast cancer cells via induction of E-cadherin expression. Our results suggest that FOXO1 3′UTR may function as a miRNA-inhibitor in modulating metastasis of breast cancer cells.     

miR-21 targets the tumor suppressor RhoB and regulates proliferation, invasion and apoptosis in colorectal cancer cells

It has become increasingly clear that microRNAs play an important role in many human diseases including cancer. Here, we show that expression of miR-21 in HEK293 and several colorectal cancer cells was found inversely correlated with ras homolog gene family, member B (RhoB) expression. miR-21 expression significantly suppressed RhoB 3' UTR luciferase-reporter activity, but the inhibitory effect was lost when the putative target sites were mutated. Exogenous miR-21 over-expression mimicked the effect of RhoB knockdown in promoting proliferation and invasion and inhibiting apoptosis, whereas anti-miR-21 or RhoB expression yielded opposite effects, in colorectal cancer cells. These results suggest that miR-21 is a regulator of RhoB expression and RhoB could be a useful target in exploring the potential therapeutic benefits of miR-21 mediated tumor cell behaviors in colorectal cancer.     

MicroRNA-22 can reduce parathymosin expression in transdifferentiated hepatocytes

Pancreatic acinar cells AR42J-B13 can transdifferentiate into hepatocyte-like cells permissive for efficient hepatitis B virus (HBV) replication. Here, we profiled miRNAs differentially expressed in AR42J-B13 cells before and after transdifferentiation to hepatocytes, using chip-based microarray. Significant increase of miRNA expression, including miR-21, miR-22, and miR-122a, was confirmed by stem-loop real-time PCR and Northern blot analyses. In contrast, miR-93, miR-130b, and a number of other miRNAs, were significantly reduced after transdifferentiation. To investigate the potential significance of miR-22 in hepatocytes, we generated cell lines stably expressing miR-22. By 2D-DIGE, LC-MS/MS, and Western blot analyses, we identified several potential target genes of miR-22, including parathymosin. In transdifferentiated hepatocytes, miR-22 can inhibit both mRNA and protein expression of parathymosin, probably through a direct and an indirect mechanism. We tested two computer predicted miR-22 target sites at the 3' UTR of parathymosin, by the 3' UTR reporter gene assay. Treatment with anti-miR-22 resulted in significant elevation of the reporter activity. In addition, we observed an in vivo inverse correlation between miR-22 and parathymosin mRNA in their tissue distribution in a rat model. The phenomenon that miR-22 can reduce parathymosin protein was also observed in human hepatoma cell lines Huh7 and HepG2. So far, we detected no major effect on several transdifferentiation markers when AR42J-B13 cells were transfected with miR-22, or anti-miR-22, or a parathymosin expression vector, with or without dexamethasone treatment. Therefore, miR-22 appears to be neither necessary nor sufficient for transdifferentiation. We discussed the possibility that altered expression of some other microRNAs could induce cell cycle arrest leading to transdifferentiation.     

The neurobiology and evolution of cannabinoid signalling

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is Delta(9)-tetrahydrocannabinol, which exerts effects in the brain by binding to a G-protein-coupled receptor known as the CB1 cannabinoid receptor. The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB1. Two putative endocannabinoid ligands, arachidonylethanolamide ('anandamide') and 2-arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system. Little is known about how or where these compounds are synthesized in the brain and how this relates to CB1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of 'classical' neurotransmitters. Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB1-expressing axon terminals. Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule to modulate neurotransmitter release from presynaptic terminals. Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility. The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non-mammalian animals. Here we review the evidence for the existence of cannabinoid receptors in non-mammalian vertebrates and invertebrates and discuss the evolution of the cannabinoid signalling system. Genes encoding orthologues of the mammalian CB1 receptor have been identified in a fish, an amphibian and a bird, indicating that CB1 receptors may occur throughout the vertebrates. Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB1 orthologues, indicating that CB1-like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes. Phylogenetic analysis of the relationship of vertebrate CB1 receptors with other G-protein-coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors. Interestingly, as with CB1, each of these receptor types does not appear to have Drosophila orthologues, indicating that this group of receptors may not occur in protostomian invertebrates. We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.     

A test of the null model for 5' UTR evolution based on GC content

Eukaryotic mRNAs are headed by a stretch of noncoding sequence,the 5' untranslated region (UTR). It has been proposed thatthe length of 5' UTRs is selectively neutral and evolves undera process of stochastic destruction and recruitment of corepromoter elements, combined with selection against the prematureinitiation of translation. We test this null model by investigatingwhether 5' UTR length varies with genomic GC content, an implicitprediction of the model. Using simulations, we show that thenull model predicts a positive relationship between GC contentand UTR length for genes regulated by a TATA box. Although thisprediction is borne out qualitatively in genomic data from yeast,fruit flies, and humans, we find marked quantitative discrepancies.We conclude that UTR length may be shaped to some degree bythe forces considered in the null model but that the model failsto provide a complete explanation for UTR length evolution.     

Increased complexity of gene structure and base composition in vertebrates

How the structure and base composition of genes changed with the evolution of vertebrates remains a puzzling question. Here we analyzed 895 orthologous protein-coding genes in six multicellular animals: human, chicken, zebrafish, sea squirt, fruit fly, and worm. Our analyses reveal that many gene regions, particularly intron and 3' UTR, gradually expanded throughout the evolution of vertebrates from their invertebrate ancestors, and that the number of exons per gene increased. Studies based on all protein-coding genes in each genome provide consistent results.We also find that GC-content increased in many gene regions (especially 5' UTR) in the evolution of endotherms, except in coding-exons.Analysis of individual genomes shows that 3′ UTR demonstrated stronger length and CC-content correlation with intron than 5' UTR, and gene with large intron in all six species demonstrated relatively similar GC-content. Our data indicates a great increase in complexity in vertebrate genes and we propose that the requirement for morphological and functional changes is probably the driving force behind the evolution of structure and base composition complexity in multicellular animal genes.     

Regulation of INSIG2 by microRNA-96

Mature forms of the microRNAs miR-96, -182, and -183 originate from a single genomic locus and have been shown to be elevated approximately 50-fold in the livers of sterol regulatory element-binding protein-1a and -2 (SREBP-1a and -2) transgenic mice. Our study attempted to identify the possible targets of these microRNAs using miRNA target prediction software. This revealed putative sites in insulin-induced genes (INSIGs). The 3′ untranslated region (UTR) of insulin-induced gene 1 (INSIG1) contained sites corresponding to miR-182, and -183, while the 3′ UTR of INSIG2 featured an miR-96 site. Among these putative sites, only miR-96 demonstrated an inhibitory effect that was specific to the 3′ UTR of INSIG2. As INSIG proteins are the main components of SREBP cleavage complexes that act to release active SREBPs, we assessed the effects of miR-96 on INSIG and SREBP levels and activities. We found that miR-96 reduced the levels of INSIG2 in INSIG1 knockout human fibroblasts, resulting in an increase in SREBP-1 and -2 nuclear forms and a subsequent increase in the abundance of the mRNA of their target genes. These results suggest that miR-96, an miRNA induced by SREBP-2 activation, regulates downstream targets of SREBPs and may increase the abundance of active SREBP.