A Rapid Evolving microRNA Cluster Rewires Its Target Regulatory Networks in Drosophila
ABSTRACT: New miRNAs are evolutionarily important but their functional evolution remains unclear. Here we report that the evolution of a microRNA cluster, mir-972C rewires its downstream regulatory networks in Drosophila. Genomic analysis reveals that mir-972C originated in the common ancestor of Drosophila where it comprises six old miRNAs. It has subsequently recruited six new members in the melanogaster subgroup after evolving for at least 50 million years. Both the young and the old mir-972C members evolved rapidly in seed and non-seed regions. Combining target prediction and cell transfection experiments, we found that the seed and non-seed changes in individual mir-972C members cause extensive target divergence among D. melanogaster, D. simulans, and D. virilis, consistent with the functional evolution of mir-972C reported recently. Intriguingly, the target pool of the cluster as a whole remains relatively conserved. Our results suggest that clustering of young and old miRNAs broadens the target repertoires by acquiring new targets without losing many old ones. This may facilitate the establishment of new miRNAs in existing regulatory networks.
Project description:MicroRNAs (miRNAs) are important posttranscriptional regulators of gene expression. However, comprehensive expression profiles of miRNAs during mammalian spermatogenesis are lacking. Herein, we sequenced small RNAs in highly purified mouse spermatogenic cells at different stages. We found that a family of X-linked miRNAs named spermatogenesis-related miRNAs (spermiRs) is predominantly expressed in the early meiotic phases and has a conserved testis-specific high expression pattern in different mammals. We identified one spermiR homolog in opossum; this homolog might originate from THER1, a retrotransposon that is active in marsupials but extinct in current placental mammals. SpermiRs have expanded rapidly with mammalian evolution and are diverged into two clades, spermiR-L and spermiR-R, which are likely to have been generated at least in part by tandem duplication mediated by flanking retrotransposable elements. Notably, despite having undergone highly frequent lineage-specific duplication events, the sequences encoding all spermiR family members are strictly located between two protein-coding genes, Slitrk2 and Fmr1. Moreover, spermiR-Ls and spermiR-Rs have evolved different expression patterns during spermatogenesis in different mammals. Intriguingly, the seed sequences of spermiRs, which are critical for the recognition of target genes, are highly divergent within and among mammals, whereas spermiR target genes largely overlap. When miR-741, the most highly expressed spermiR, is knocked out in cultured mouse spermatogonial stem cells (SSCs), another spermiR, miR-465a-5p, is dramatically upregulated and becomes the most abundant miRNA. Notably, miR-741-/- SSCs grow normally, and the genome-wide expression levels of mRNAs remain unchanged. All these observations indicate functional compensation between spermiR family members and strong coevolution between spermiRs and their targets.
Project description:MicroRNAs (miRNAs) regulate the expression of mRNA through sequence-specific binding of the 3' untranslated region (UTR). The seed sequence of miRNAs is the key determinant for target site recognition. Paralogous miRNAs, which share the same seed sequences but differ in their 3' regions, are known to regulate largely overlapping groups of mRNAs. However, no study has analyzed functional differences between paralogous miRNAs with proper experimental methods. In this study, we compared the targets of paralogous miRNAs, miR-221 and miR-222. Using a nuclease-mediated genome engineering technique, we established knockout cell lines for these miRNAs, and precisely analyzed differences in target regulation. We found that miR-221 and miR-222 suppress the previously identified targets, CDKN1B and CDKN1C, differentially. Whereas both miRNAs suppressed CDKN1B, only miR-221 suppressed CDKN1C. From transcriptome analyses, we found that several different target mRNAs were regulated by each of miR-221 and miR-222 independently, although a large number of mRNAs responded commonly to miR-221 and miR-222. This is the first study to compare the mRNA regulations by paralogous miRNAs and illustrate that paralogous miRNAs with the same seed sequence also have difference in target regulation.
Project description:Expression of the mouse miR-290-295 cluster and its miR-371-373 homolog in human is restricted to early embryos, primordial germ cells, the germ line stem cell compartment of the adult testis and to stem cell lines derived from the early embryonic lineages. Sequencing data suggest considerable seed diversification between the seven homologous pre-miRNAs of miR-290-295 but it is not clear if all of the implied miR-290-295 seeds are also conserved in the human miR-371-373 cluster, which consists of only three homologous pre-miRNAs. By employing miRNA target reporters we show that most, if not all, seeds in miR-290-295 are represented in miR-371-373. In the mouse, pre-miR-290, pre-miR-292 and pre-miR-293 express subsets of the miRNA isoforms processed from the single human pre-miR-371. Comparison of the possible miR-290-295/miR-371-373 seed repertoires in placental mammals suggests a model for the evolution of this miRNA cluster family, which would be otherwise difficult to deduce based solely on pre-miRNA sequence comparisons. The conservation of co-expressed seeds that is characteristic of miR-290-295/miR-371-373 should be taken into account in models of the corresponding miRNA-target interaction networks.
Project description:microRNAs (miRNAs) repress target genes by destabilizing mRNAs and/or by inhibiting translation. The best known factor for target recognition is the so called seed--a short continuous region of Watson-Crick base pairing between nucleotides 2-7 of the miRNA and complementary sequences in 3' untranslated regions of target mRNAs. The miR-34 family consists of three conserved members with important tumor suppressor functions linked to the p53 pathway. The family members share the same seed, raising the question if they also have the same targets. Here, we analyse the effect of miR-34a and miR-34c on protein synthesis by pSILAC. Despite significant overlap, we observe that the impact of both family members on protein synthesis differs. The ability to identify specific targets of a family member is complicated by the occurrence of * strand mediated repression. Transfection of miR-34 chimeras indicates that the 3'end of the miRNA might be responsible for differential regulation in case of targets without a perfect seed site. Pathway analysis of regulated proteins indicates overlapping functions related to cell cycle and the p53 pathway and preferential targeting of several anti-apoptotic proteins by miR-34a. We used luciferase assays to confirm that Vcl and Fkbp8, an important anti-apoptotic protein, are specifically repressed by miR-34a. In summary, we find that miR-34a and miR-34c down-regulate distinct subsets of targets which might mediate different cellular outcomes. Our data provides a rich resource of miR-34 targets that might be relevant for clinical trials that want to implement the miR-34 family in cancer therapy.
Project description:MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host–virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 59 end of miRNA. The 59 ends of the miRNAs were more conserved than the 39 ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect’s miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host–virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs. Identification of miRNA and other small non coding RNA in NPV infected Sf9 cells
Project description:<h4>Background</h4>microRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies.<h4>Results</h4>We report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages.<h4>Conclusion</h4>This study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.
Project description:The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.
Project description:The role of miRNAs in mediating insecticide resistance remains largely unknown, even for the model species Drosophila melanogaster. Building on prior research, this study used microinjection of synthetic miR-310s mimics into DDT-resistant 91-R flies and observed both a significant transcriptional repression of computationally-predicted endogenous target P450 detoxification genes, Cyp6g1 and Cyp6g2, and also a concomitant increase in DDT susceptibility. Additionally, co-transfection of D. melanogaster S2 cells with dual luciferase reporter constructs validated predictions that miR-310s bind to target binding sites in the 3' untranslated regions (3'-UTR) of both Cyp6g1 and Cyp6g2 in vitro. Findings in the current study provide empirical evidence for a link between reduced miRNA expression and an insecticidal resistance phenotype through reduced targeted post-transcriptional suppression of transcripts encoding proteins involved in xenobiotic detoxification. These insights are important for understanding the breadth of adaptive molecular changes that have contributed to the evolution of DDT resistance in D. melanogaster.
Project description:MicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.
Project description:Somatic cells can be reprogrammed to an ES-like state to create induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and cMyc. Here, we show that cellular microRNAs (miRNAs) regulate iPSC generation. Knock-down of key microRNA pathway proteins resulted in significant decreases in reprogramming efficiency. Three miRNA clusters, miR-17?92, miR-106b?25 and miR-106a?363, were shown to be highly induced during early reprogramming stages. Several miRNAs, including miR-93 and miR-106b, which have very similar seed regions, greatly enhanced iPSC induction and modulated mesenchymal-to-epithelial transition step in the initiation stage of reprogramming, and inhibiting these miRNAs significantly decreased reprogramming efficiency. Moreover, miR-iPSC clones reached the fully reprogrammed state. Further analysis revealed that Tgfbr2 and p21 are directly targeted by these miRNAs and that siRNA knock-down of both genes indeed enhanced iPSC induction. Here, for the first time, we demonstrate that miR-93 and its family members directly target TGF-? receptor II to enhance iPSC generation. Overall, we demonstrate that miRNAs function in the reprogramming process and that iPSC induction efficiency can be greatly enhanced by modulating miRNA levels in cells.