Analysis of the regulation of lin-41 during chick and mouse limb development.

We have cloned the chicken and mouse orthologues of the Caenorhabditis elegans heterochronic gene lin-41. During limb development, lin-41 is expressed in three phases over developmental time and most notably is associated with the developing autopod. Using chicken and mouse mutants and bead implantations, we report that lin-41 is genetically and biochemically downstream of both the Shh and Fgf signaling pathways. In C. elegans, it is proposed that lin-41 activity is temporally regulated by miRNAs (let-7 and lin-4) that bind to complementary sites in the lin-41 3'-untranslated region (UTR). Taking a bioinformatics approach, we also report the presence of potential miRNA binding sites in the 3'-UTR of chicken lin-41, including sites for the chicken orthologues of both C. elegans let-7 and lin-4. Finally, we show that these miRNAs and others are expressed in the chick limb consistent with the hypothesis that they regulate chicken Lin-41 activity in vivo.     

Cloning and regulation of the vertebrate homologue of lin-41 that functions as a heterochronic gene in Caenorhabditis elegans.

The Caenorhabditis elegans gene lin-41 is the one of the heterochronic genes that regulate the timing of many developmental events. MicroRNA let-7 negatively regulates the expression of lin-41 through RNA-RNA interaction on its 3' untranslated region (UTR). Here, we report the isolation of chick and mouse homologues of lin-41 that encode the RBCC-NHL family protein and their expression patterns. C. elegans lin-41 is one of the RBCC-NHL families and the predicted amino acid sequences of isolated two genes encode the same family proteins. Chick and mouse lin-41 expression was also observed in developing limb buds, branchial arches, and tail buds. The 3'UTRs of the mouse and chick lin-41 genes contain multiple let-7 complementary sites. Using luciferase assay, we showed that lin-41 expression can be regulated through let-7 complementary sites.     

Post-embryonic expression of C. elegans microRNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system.

MicroRNAs (miRNAs) are regulatory molecules that negatively control gene expression by binding to complementary sequences on target mRNAs. The most thoroughly characterized miRNAs, lin-4 and let-7, direct cell fate determination during the larval transitions in C. elegans and act as key regulators of temporal gene expression. lin-4 and let-7 are founding members of two distinct families of miRNA genes sharing strong sequence homology primarily in the 5' end of the mature miRNAs. In this report, we characterize the temporal and spatial expression patterns of lin-4 and let-7 family members using northern blot analysis and mir::gfp fusion studies. Our results show that lin-4 and let-7 homologues possess distinct temporal and spatial expression patterns during nematode development and that known heterochronic genes regulate their expression. We find that certain lin-4 and let-7 family members display overlapping expression patterns in the hypodermis and the reproductive system, suggesting that combinations of miRNAs from across families may control common developmental events.     

The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3'UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3'UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3'UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.     

Drosophila let-7 microRNA is required for remodeling of the neuromusculature during metamorphosis

The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). We find that the let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. We find that the function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-to-adult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which let-7-C promotes adult behavior.     

'Inc-miRs': functional intron-interrupted miRNA genes

The discovery of microRNAs (miRNAs) lin-4 and let-7 as temporal regulators in Caenorhabditis elegans led to broader searches for novel miRNAs and their biological roles. Unlike protein-coding genes and some long noncoding RNAs, canonical metazoan miRNAs are not known to contain introns within their genomic precursor sequences. Because the short length of miRNAs complicates a statistically definitive assignment of split genes in RNA sequencing data sets, we took an experimental approach toward testing the compatibility of splicing and functional miRNA biogenesis. To definitively evaluate the possibility that miRNAs could derive from interrupted genes, we constructed intron-interrupted variants of C. elegans lin-4 and assayed for their miRNA-encoding capability and biological activity in the developing organism. Our studies indicate that (1) intron-containing miRNAs (inc-miRs) can be efficiently spliced and processed to produce miRNAs with normal termini, and (2) these miRNAs can be functional in full rescue of developmental phenotypes in null mutants lacking endogenous lin-4. This study provides the first evidence to support the ability of intron-interrupted miRNA precursors to produce functional regulators and identifies an additional modality available for metazoan miRNA production.     

The microRNAs of Caenorhabditis elegans

MicroRNAs (miRNAs) are an abundant class of tiny RNAs thought to regulate the expression of protein-coding genes in plants and animals. In the present study, we describe a computational procedure to identify miRNA genes conserved in more than one genome. Applying this program, known as MiRscan, together with molecular identification and validation methods, we have identified most of the miRNA genes in the nematode Caenorhabditis elegans. The total number of validated miRNA genes stands at 88, with no more than 35 genes remaining to be detected or validated. These 88 miRNA genes represent 48 gene families; 46 of these families (comprising 86 of the 88 genes) are conserved in Caenorhabditis briggsae, and 22 families are conserved in humans. More than a third of the worm miRNAs, including newly identified members of the lin-4 and let-7 gene families, are differentially expressed during larval development, suggesting a role for these miRNAs in mediating larval developmental transitions. Most are present at very high steady-state levels-more than 1000 molecules per cell, with some exceeding 50,000 molecules per cell. Our census of the worm miRNAs and their expression patterns helps define this class of noncoding RNAs, lays the groundwork for functional studies, and provides the tools for more comprehensive analyses of miRNA genes in other species.     

A matter of timing: microRNA-controlled temporal identities in worms and flies

The first microRNAs were identified in Caenorhabditis elegans based on their functions in the temporal regulation of stage-specific cell fate decisions. Until now, it was not known whether the so-called heterochronic genes that encode miRNAs are also involved in controlling developmental transitions in other organisms. New findings by Sokol et al. (this issue of Genes & Development, pp. 1591-1596) demonstrate that the Drosophila counterpart of a heterochronic miRNA gene from C. elegans, let-7, does indeed play a role in promoting stage-specific developmental events in neuromuscular tissues during the transition from larval to adult stages, thus pointing to a more widespread utilization of miRNAs in temporal regulation of animal development.     

Temporal regulation of lin-14 by the antagonistic action of two other heterochronic genes, lin-4 and lin-28

Heterochronic genes form a regulatory pathway that controls the temporal sequence of the Caenorhabditis elegans postembryonic cell lineage. One of these genes, lin-14, encodes a nuclear protein that constitutes a temporal developmental switch. During wild-type development, lin-14 protein is abundant during early larval stage 1 (L1) to specific L1-specific cell lineages but is nearly undetectable at L2 and later stages to specify L2-specific and later cell lineages. To determine the roles played by other genes in executing this temporal switch, we have analyzed how lin-14 expression is regulated by other heterochronic genes. lin-4 is required to down-regulate lin-14 protein levels during the L1 stage, whereas lin-28 positively regulates lin-14 protein levels. The lin-4 gene product is a candidate for interacting with the negative regulatory element in the 3'-untranslated region of lin-14. lin-29 mutations do not affect lin-14 protein levels, consistent with lin-29 acting downstream of lin-14. Switching off lin-14 expression during the L1 stage is not triggered by the passage of time per se but, rather, is normally dependent on feeding or the feeding-dependent initiation of postembryonic cell division.     

A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas

Human uterine leiomyomas (ULMs) are the most common neoplasms of women. Many genes are dysregulated in ULMs and some of this dysregulation may be due to abnormal expression of micro-RNAs (miRNAs). In this study, 55 ULMs and matched myometrium were collected from 41 patients for microarray-based global miRNA expression analysis. Of 206 miRNAs examined, 45 miRNAs were significantly up- or down-regulated in ULMs in comparison to the matched myometrium (P < 0.001). The top five dysregulated miRNAs in ULMs are the let-7 family, miR-21, miR-23b, miR-29b, and miR-197. Four polycistronic clusters of miRNAs were either up- or down-regulated, but not in a mixed pattern, indicative of coordinated regulation of these miRNAs. Significance analysis revealed that subsets of miRNAs were strongly associated with tumor sizes and race. By prediction analysis we identified some important tumorigenic genes previously identified in ULMs that may be targeted by the dysregulated miRNAs. HMGA2 was identified as one of target genes of the let-7 family of miRNAs and has been found to be suppressed by let-7 in vitro. This article contains Supplementary material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Systematic discovery and characterization of fly microRNAs using 12 Drosophila genomes

MicroRNAs (miRNAs) are short regulatory RNAs that inhibit target genes by complementary binding in 3' untranslated regions (3' UTRs). They are one of the most abundant classes of regulators, targeting a large fraction of all genes, making their comprehensive study a requirement for understanding regulation and development. Here we use 12 Drosophila genomes to define structural and evolutionary signatures of miRNA hairpins, which we use for their de novo discovery. We predict >41 novel miRNA genes, which encompass many unique families, and 28 of which are validated experimentally. We also define signals for the precise start position of mature miRNAs, which suggest corrections of previously known miRNAs, often leading to drastic changes in their predicted target spectrum. We show that miRNA discovery power scales with the number and divergence of species compared, suggesting that such approaches can be successful in human as dozens of mammalian genomes become available. Interestingly, for some miRNAs sense and anti-sense hairpins score highly and mature miRNAs from both strands can indeed be found in vivo. Similarly, miRNAs with weak 5' end predictions show increased in vivo processing of multiple alternate 5' ends and have fewer predicted targets. Lastly, we show that several miRNA star sequences score highly and are likely functional. For mir-10 in particular, both arms show abundant processing, and both show highly conserved target sites in Hox genes, suggesting a possible cooperation of the two arms, and their role as a master Hox regulator.     

Characterization of microRNA profile in IgE-mediated mouse BMMCs degranulation

BackgroundMast cells play a central role in innate and adaptive immunity by releasing pre-formed and de novo synthesized mediators, which include microRNAs. Although miRNAs have been confirmed to function in cell proliferation, differentiation, apoptosis, and the immune response, their functions are still limited in mast cells degranulation.MethodsHere, we survey miRNA expression profiles in activated mouse bone marrow-derived mast cells (BMMCs) with a miRNA microarray and compare the profiles to those from resting BMMCs. Partial miRNAs were selected for confirmation by qPCR, and let-7i was selected for function discover in mast cell degranulation process. TargetScan Mouse database were used for target genes prediction, gene ontology (GO) were used for gene molecular function classifications, and Cytoscape software were used to construct gene network of degranulation.ResultsWe found 13 up-regulated miRNAs and 7 down-regulated miRNAs in DNP activated BMMCs by miRNA microarray; and let-7b, let-7c, let-7d, let-7f, let-7i, and miR-652 were up-regulated, and miR-296-3p was down-regulated in DNP-stimulated BMMCs by qPCR. In the function research, let-7i can inhibit mast cell degranulation by suppress Exco8 expression. Overall, the data indicate that miRNAs participate in mast cell activation, especial for mast cell degranulation process.     

microRNA expression profiling of the developing mouse heart

microRNAs (miRNAs) play an important role in regulating normal organ physiology and development. Many miRNAs show spatially and temporally restricted expression patterns during embryogenesis and organogenesis. This study aimed to characterize the miRNA profile of the fetal mouse heart at 4 key time-points [embryonic day (E)12.5, E14.5, E16.5 and E18.5] in its development, by performing a sequencing by oligonucleotide ligation and detection (SOLiD) miRNA screen. The 4 time-points were designated as groups M1 (E18.5), M2 (E16.5), M3 (E14.5) and M4 (E12.5). miRNAs found to have consistent fold-changes of >2.0) between the 4 time-points were selected for further analysis. Ten miRNAs (mmu-miR-23b, mmu-miR-24, mmu-miR-23a, mmu-miR-375, mmu-miR-29a, mmu-miR-93, mmu-miR-21, mmu-miR-25, mmu-let-7b and mmu-miR-27b) that were the most highly expressed in the 4 groups, including the percentage >1% of total read counts, were identified. No miRNA was consistently downregulated or upregulated. There were 16 differentially expressed miRNAs between the later development group (M1+M2) and the early development group (M3+M4), which were validated by quantitative real-time PCR. Several members of the let-7 miRNA cluster (mmu-let-7a/7d/7e/7f) were upregulated in the later development group compared with the early development group. A network analysis of the predicted targets of mmu-let-7a/7d/7e/7f identified 5 target genes (FOXP1, TBX5, HAND1, AKT2 and PPARGC1A), known to be involved in cardiac development. Therefore, this study identified several miRNAs that are abundantly expressed in the developing heart, several of which are differentially expressed in the 4 time-points studied. Findings of this analysis may thus clarify the mechanisms of normal heart development and provide a physiological basis for future studies on congenital heart disease.     

Prediction of conserved microRNAs from skin and mucosal human papillomaviruses

Eight human papillomavirus (HPV) types including four cutaneous HPV types (HPV-5, HPV-8, HPV-20 and HPV-38) and four mucosal HPV types (HPV-6, HPV-11, HPV-16 and HPV-18) were selected for this miRNA study. Pre-miRNAs were predicted using a computer programme, and the conserved mature miRNAs were compared to currently known miRNAs. Predicted HPV miRNAs related to miR-466, -467 and -669 were common and specific to the mucosal HPV types. Northern blot hybridization confirmed a predicted miRNA in HPV-positive cervical cancer cell lines encoded by mucosal HPVs. HPV-38 was predicted to express an miRNA conserved to human let-7a and the expression of let-7a, in HPV-38-positive non-melanoma skin cancer (NMSC) biopsies was 10-fold higher than those with HPV-positive (for other types except HPV-38) and HPV-negative NMSCs, suggesting that let-7a expression might be related to HPV-38 infection. Potential gene targets of the predicted miRNA that may aid HPV in infection and pathogenesis were also analysed.