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Results for “MicroRNA, (miRNA)”

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PubMed2002

miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs.

Gemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large approximately 15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem-loop structures.

Animals↗

PubMed2005

The widespread impact of mammalian MicroRNAs on mRNA repression and evolution.

Thousands of mammalian messenger RNAs are under selective pressure to maintain 7-nucleotide sites matching microRNAs (miRNAs). We found that these conserved targets are often highly expressed at developmental stages before miRNA expression and that their levels tend to fall as the miRNA that targets them begins to accumulate. Nonconserved sites, which outnumber the conserved sites 10 to 1, also mediate repression. As a consequence, genes preferentially expressed at the same time and place as a miRNA have evolved to selectively avoid sites matching the miRNA. This phenomenon of selective avoidance extends to thousands of genes and enables spatial and temporal specificities of miRNAs to be revealed by finding tissues and developmental stages in which messages with corresponding sites are expressed at lower levels.

Animals↗

PubMed2006

miTarget: microRNA target gene prediction using a support vector machine.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs, which play significant roles as posttranscriptional regulators. The functions of animal miRNAs are generally based on complementarity for their 5' components. Although several computational miRNA target-gene prediction methods have been proposed, they still have limitations in revealing actual target genes. RESULTS: We implemented miTarget, a support vector machine (SVM) classifier for miRNA target gene prediction. It uses a radial basis function kernel as a similarity measure for SVM features, categorized by structural, thermodynamic, and position-based features. The latter features are introduced in this study for the first time and reflect the mechanism of miRNA binding. The SVM classifier produces high performance with a biologically relevant data set obtained from the literature, compared with previous tools. We predicted significant functions for human miR-1, miR-124a, and miR-373 using Gene Ontology (GO) analysis and revealed the importance of pairing at positions 4, 5, and 6 in the 5' region of a miRNA from a feature selection experiment. We also provide a web interface for the program. CONCLUSION: miTarget is a reliable miRNA target gene prediction tool and is a successful application of an SVM classifier. Compared with previous tools, its predictions are meaningful by GO analysis and its performance can be improved given more training examples.

Algorithms↗

PubMed2006

MicroRNA involvement in mammary gland development and breast cancer.

MicroRNA (miRNA) are small non-coding RNA that post-transcriptionally regulate gene expression. In humans, miRNA genes may account for 2 to 3% of the total number of genes. Although the biological functions of most miRNA are unknown, their importance for development, cell proliferation, cell death, and morphogenesis has been demonstrated in several species. One could thus speculate that miRNA should be involved in the regulation of one of the organs that can undergo cycles of cell division, differentiation and dedifferentiation in the adult, the mammary gland. In this paper we summarise several reports dealing with the potential implication of miRNA in the mammary gland, most of them focussed on pathological situations, such as the appearance of breast cancer. These data suggest an implication of miRNA on mammary gland biology. However, direct evidence of this is still lacking. Expression profile analysis of miRNA during the normal mammary gland development could help in addressing this question and in identifying miRNA potentially involved. To this aim, we undertook such an analysis on mouse mammary gland at different stages (virgin, pregnancy, lactation and involution) and will present our preliminary results.

Animals↗

PubMed2006

The colorectal microRNAome.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.

Base Sequence↗

PubMed2006

Effect of adenovirus-mediated RNA interference on endogenous microRNAs in a mouse model of multidrug resistance protein 2 gene silencing.

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein.

microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs(f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.

Alternative Splicing↗

PubMed2026

MicroRNAs and predicted targets in the switch from monolayered to spheroids of cholangiocarcinoma cells.

BACKGROUND: Extrahepatic cholangiocarcinoma (eCCA) is characterized by marked molecular heterogeneity and limited therapeutic options. MicroRNAs (miRNAs) are key post-transcriptional regulators of cancer-related pathways, but their contribution to tumor adaptation in physiologically relevant models remains poorly understood. Three-dimensional (3D) tumor spheroids better mimic in vivo conditions than conventional two-dimensional (2D) cultures. METHODS: We compared miRNA expression profiles in two eCCA cell lines (Sk-ChA-1 and Mz-ChA-1) grown as monolayers (2D) or multicellular tumor spheroids (3D). MiRNA profiling was performed using NanoString technology. Predicted targets were analyzed by over-representation analysis, and selected miRNAs and genes were validated by RT-qPCR and ELISA-based assays. RESULTS: 3D growth induced extensive miRNA remodeling, with distinct (54 deregulated in Sk-ChA-1 and 29 in Mz-ChA-1 cells) and partially overlapping signatures (miR-1283, miR-577, and miR-2113). Among the shared miRNAs, predicted targets included DUSP10 and RBFOX1, while in spheroids, cell-specific multiple miRNAs converged on shared targets (TNRC6B, SMARCAD1, ATG14, HMGA2, and CLOCK) displaying inverse expression patterns. The transcriptional program impacted MAPK signaling, enhanced EMT, and activated stress-adaptive networks but attenuated proliferation in 3D Sk-ChA-1 cells, while Mz-ChA-1 cells retained a more epithelial and proliferative profile. In this context, we point out the involvement of miR-19b-3p using anti-miR transfection experiments. CONCLUSION: Our findings reveal a miRNA-driven regulatory landscape associated with 3D growth in eCCA, linking tumor architecture to signaling rewiring and cellular plasticity, and highlight potentially druggable candidate targets and pathways to investigate as candidates using inhibitors or gene therapy-based interventions.

Humans↗

PubMed2005

Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals.

Comprehensive identification of all functional elements encoded in the human genome is a fundamental need in biomedical research. Here, we present a comparative analysis of the human, mouse, rat and dog genomes to create a systematic catalogue of common regulatory motifs in promoters and 3' untranslated regions (3' UTRs). The promoter analysis yields 174 candidate motifs, including most previously known transcription-factor binding sites and 105 new motifs. The 3'-UTR analysis yields 106 motifs likely to be involved in post-transcriptional regulation. Nearly one-half are associated with microRNAs (miRNAs), leading to the discovery of many new miRNA genes and their likely target genes. Our results suggest that previous estimates of the number of human miRNA genes were low, and that miRNAs regulate at least 20% of human genes. The overall results provide a systematic view of gene regulation in the human, which will be refined as additional mammalian genomes become available.

3' Untranslated Regions↗

PubMed2006

Characterization of five microRNA families in maize.

In recent years, microRNAs (miRNAs) have polarized the interest of the scientific community as a new category of gene expression regulators, present in both plants and animals. Plant miRNAs are involved in processes such as plant development, organ identity, and stress response. Nonetheless, knowledge of their functions is still incomplete, and it is conceivable that further new processes in which they are involved will be discovered. For these reasons, structural and functional characterization of MIR genes, that are also in crop species such as Zea mays L., becomes instrumental in addressing genetic and molecular mechanisms controlling phenotype determination and phenotypic adaptation to growing conditions. The present study contributes to the characterization of five miRNA families in maize, from the determination of their expression pattern in different maize tissues and genotypes, to the identification of putative targets by bioinformatic means and subsequent experimental validation of three targets by modified 5' RACE experiments. Furthermore, 30 different MIR genes belonging to these five miRNA families were analysed by their attribution to maize chromosomes using oat-maize addition lines and by investigating their phylogenetic relationship with genes from other cereals. In particular, sequence homology was determined by the reciprocal best BLAST hit approach, to define groups of homologous genes between maize, rice, and sorghum.

Chromosome Mapping↗

PubMed2005

MicroRNAs: critical regulators of development, cellular physiology and malignancy.

MicroRNAs (miRNAs) are 18-24 nucleotide RNA molecules that regulate the stability or translational efficiency of target mRNAs. These regulatory RNAs function by acting as sequence-specific guides which recruit a large protein complex known as the RNA-induced silencing complex, or RISC, to target mRNAs which are subsequently silenced. Diverse functions have been attributed to miRNAs including the regulation of cellular differentiation, proliferation, and apoptosis. Moreover, significant evidence has accumulated implicating a fundamental role for miRNAs in the development of cancer. We recently demonstrated that the oncogenic transcription factor c-Myc regulates a group of miRNAs known as the mir-17 cluster. This represents the first documented example of a mammalian transcription factor that regulates miRNA expression. Moreover, it was independently demonstrated that the mir-17 cluster accelerates c-Myc-induced lymphomagenesis in an in vivo mouse model. Together, these studies support an important role for this group of miRNAs in c-Myc-mediated tumorigenesis. We have also demonstrated that two miRNAs in this cluster regulate the pro-proliferative, pro-apoptotic transcription factor E2F1. Herein, we propose a model in which the mir-17 cluster prevents excessive E2F1 activity, and thereby apoptosis, in response to activation of c-Myc.

3' Untranslated Regions↗

PubMed2005

Identification of microRNAs of the herpesvirus family.

Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.

Chromosome Mapping↗

PubMed2004

An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues.

MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissue-specific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.

Adult↗

PubMed2004

Quantitation of microRNAs using a modified Invader assay.

The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.

Base Sequence↗

PubMed2005

MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.

Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.

3' Untranslated Regions↗

PubMed2006

ProMiR II: a web server for the probabilistic prediction of clustered, nonclustered, conserved and nonconserved microRNAs.

ProMiR is a web-based service for the prediction of potential microRNAs (miRNAs) in a query sequence of 60-150 nt, using a probabilistic colearning model. Identification of miRNAs requires a computational method to predict clustered and nonclustered, conserved and nonconserved miRNAs in various species. Here we present an improved version of ProMiR for identifying new clusters near known or unknown miRNAs. This new version, ProMiR II, integrates additional evidence, such as free energy data, G/C ratio, conservation score and entropy of candidate sequences, for more controllable prediction of miRNAs in mouse and human genomes. It also provides a wider range of services, e.g. the prediction of miRNA genes in long nonrelated sequences such as viral genomes. Importantly, we have validated this method using several case studies. All data used in ProMiR II are structured in the MySQL database for efficient analysis. The ProMiR II web server is available at http://cbit.snu.ac.kr/~ProMiR2/.

Animals↗

PubMed2006

The role of PACT in the RNA silencing pathway.

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.

Argonaute Proteins↗

PubMed2006

A microRNA expression signature of human solid tumors defines cancer gene targets.

Small noncoding microRNAs (miRNAs) can contribute to cancer development and progression and are differentially expressed in normal tissues and cancers. From a large-scale miRnome analysis on 540 samples including lung, breast, stomach, prostate, colon, and pancreatic tumors, we identified a solid cancer miRNA signature composed by a large portion of overexpressed miRNAs. Among these miRNAs are some with well characterized cancer association, such as miR-17-5p, miR-20a, miR-21, miR-92, miR-106a, and miR-155. The predicted targets for the differentially expressed miRNAs are significantly enriched for protein-coding tumor suppressors and oncogenes (P < 0.0001). A number of the predicted targets, including the tumor suppressors RB1 (Retinoblastoma 1) and TGFBR2 (transforming growth factor, beta receptor II) genes were confirmed experimentally. Our results indicate that miRNAs are extensively involved in cancer pathogenesis of solid tumors and support their function as either dominant or recessive cancer genes.

Gene Expression Profiling↗