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PubMed2006

Construction of microRNA-containing vectors for expression in mammalian cells.

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression by single-stranded RNAs of 18 to 25 nucleotides in length. Hundreds of miRNAs have been found in animals and plants, some of which play important roles in development or differentiation. Increasing attention has thus been paid to their biogenesis and regulation mechanisms and the identification of target genes. We are constructing a comprehensive expression vector library containing predicted human miRNAs. miRNA expression vectors containing human RNA polymerase II or III promoters, and utilizing a flexible vector system, can be useful for functional analysis.

Base Sequence↗

PubMed2006

A novel method to monitor the expression of microRNAs.

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with important roles in the regulation of gene expression. Although a large number of miRNAs have been identified in a variety of eukaryotic systems, the function of the vast majority of these molecules remains unknown. To study the functions of miRNAs, it is crucial to determine their spatial and temporal expression patterns. Although there are some existing methods that can analyze the expression of miRNAs, it is not an easy task for routine gene-expression studies. In this study, we have established a simple method to detect the expression of mature miRNAs. Total RNA was polyadenylated by poly(A) polymerase, and then cDNA was synthesized by a specific reverse transcriptase (RT) primer and reverse transcriptase using the poly(A)-tailed total RNA as templates. The expression of several mature miRNAs was assayed by this method. The expression profile of two miRNAs, determined by the polymerase chain reaction (PCR) assay, was identical to that determined by Northern blotting. All these data show that the poly(A)-tailed RT-PCR is a convenient method to detect the expression of miRNAs.

Blotting, Northern↗

PubMedDate not supplied

The emerging world of microRNAs.

MicroRNAs (miRNAs) are a family of naturally occurring, evolutionary conserved, small (approximately 19-23 nucleotides), non-protein-coding RNA molecules that generally negatively regulate post-transcriptional gene expression. miRNAs are estimated to account for >3% of all human genes and to control expression of thousands of target mRNAs, with multiple miRNAs targeting each mRNA. A role for miRNAs has been identified in both normal physiological and pathological conditions, including metabolism, proliferation, cell death, differentiation and development, insulin secretion from pancreatic beta cells, viral infection and cancer. Antisense technologies have been successfully used to control miRNA expression in vitro and in vivo. Further analysis of this interesting class of small RNAs, in normal and pathological conditions, will enable us determine their potential to be exploited as therapeutic targets in disease.

MicroRNAs↗

PubMed2006

Molecular therapy in the microRNA era.

MicroRNAs (miRNAs) consist of a growing class of non-coding RNAs (ncRNAs) that negatively regulate the expression of genes involved in development, differentiation, proliferation, apoptosis and other important cellular processes. miRNAs are usually 18-25 nt long and are each able to regulate several mRNAs by mechanisms such as incomplete base pairing and Post-Transcriptional Gene Silencing (PTGS). A growing number of reports have shown that aberrant miRNA expression is a common feature of human diseases including cancer, which has sparked interest in targeting these regulators of gene expression as a means of ameliorating these diseases. Here, we review important aspects of miRNA function in normal and pathological states and discuss new modalities of epigenetic intervention strategies that could be used to amend defects in miRNA/mRNA interactions.

Epigenesis, Genetic↗

PubMed2004

The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation.

The Arabidopsis HYL1 gene encodes a nuclear double-stranded RNA-binding protein. A knockout mutation of the hyl1 gene is recessive and pleiotropic, causing developmental abnormalities, increasing sensitivity to abscisic acid, and reducing sensitivity to auxin and cytokinin. We report that levels of several microRNAs (miRNAs; miR159, -167, and -171) are reduced in homozygous mutant plants, and levels of two of three tested target mRNAs are elevated. Conversely, the miRNA levels are elevated in plants expressing a HYL1 cDNA from a strong promoter, and the corresponding target RNAs are reduced. These changes result from alterations in the stability of the target RNAs. However, double-stranded RNA-induced posttranscriptional gene silencing is unaffected by the hyl1 mutation. One-third to one-half of the cellular HYL1 protein is in a macromolecular complex, and a GFP-HYL1 fusion protein is found predominantly in the nucleus, although it is observed in both nucleus and cytoplasm in some cells. Within nuclei, HYL1 is associated with subnuclear bodies and ring-like structures. These observations provide evidence that the HYL1 protein is part of a nuclear macromolecular complex that is involved in miRNA-mediated gene regulation. Because hyl1 mutants show marked abnormalities in hormone responses, these results further suggest that miRNA-mediated changes in mRNA stability play a vital role in plant hormone signaling.

Arabidopsis↗

PubMed2007

The MicroRNA let-7a modulates interleukin-6-dependent STAT-3 survival signaling in malignant human cholangiocytes.

The inflammation-associated cytokine interleukin-6 (IL-6) can contribute to tumor growth and resistance to therapy by the activation of survival mechanisms. In several human cancers, IL-6-activated survival signaling involves the signal transducers and activators of transcription (Stat) factors or protein kinase cascades. microRNAs (miRNAs) are endogenous regulators of gene expression that are altered in expression in many cancers. However, the effect of inflammatory cytokines on miRNA expression and the role of miRNA in modulating IL-6-mediated cell survival are unknown. We investigated the involvement of miRNA in malignant cholangiocytes stably transfected to overexpress IL-6, which enhances tumor growth in vivo by inhibition of apoptosis. We provide evidence that (i) miRNA expression both in vitro and in vivo is altered by overexpression of IL-6; (ii) selective miRNAs including let-7a are up-regulated and contribute to the survival effects of enforced IL-6 activity; and (iii) let-7a contributes to the constitutively increased phosphorylation of Stat-3 by a mechanism involving the neurofibromatosis 2 (NF2) gene. These findings reveal a novel mechanism by which IL-6 mediates tumor cell survival that may be therapeutically targeted and emphasize the presence of complex interrelationships between deregulated expression of miRNA and transcription factors in human cancers.

Adenoma, Bile Duct↗

PubMed2005

Physiological identification of human transcripts translationally regulated by a specific microRNA.

One mechanism by which endogenous microRNAs (miRNAs) function is to suppress translation of target mRNAs. Computational identification of target mRNAs is hampered by the partial complementarity between miRNAs and their targets and the lack of in vivo approaches to identify targets. Here, we identify mRNAs that are regulated by specific endogenous miRNA by detecting shifts in individual mRNA abundance in polyribosome profiles following miRNA knockdown via siRNA. We have identified human genes whose mRNAs were found at significantly increased levels in the heavy polyribosome fractions following miRNA miR-30a-3p knockdown. If antibody was available, targets showed an increase in protein levels following the miRNA knockdown and a decrease following the miRNA overexpression. Although all identified transcripts have sequences that partially complement miR-30a-3p, none was identified by commonly used computational means. These data suggest that the functional interaction between miRNAs and mRNA targets is more complex than previously realized and describe an approach to refine predictive algorithms.

3' Untranslated Regions↗

PubMed2006

Cloning and expression of new microRNAs from zebrafish.

MicroRNAs (miRNAs) play an important role in development and regulate the expression of many animal genes by post-transcriptional gene silencing. Here we describe the cloning and expression of new miRNAs from zebrafish. By high-throughput sequencing of small-RNA cDNA libraries from 5-day-old zebrafish larvae and adult zebrafish brain we found 139 known miRNAs and 66 new miRNAs. For 65 known miRNAs and for 11 new miRNAs we also cloned the miRNA star sequence. We analyzed the temporal and spatial expression patterns for 35 new miRNAs and for 32 known miRNAs in the zebrafish by whole mount in situ hybridization and northern blotting. Overall, 23 of the 35 new miRNAs and 30 of the 32 known miRNAs could be detected. We found that most miRNAs were expressed during later stages of development. Some were expressed ubiquitously, but many of the miRNAs were expressed in a tissue-specific manner. Most newly discovered miRNAs have low expression levels and are less conserved in other vertebrate species. Our cloning and expression analysis indicates that most abundant and conserved miRNAs in zebrafish are now known.

Animals↗

PubMed2006

A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.

To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.

Arabidopsis↗

PubMed2006

Bioinformatic discovery of microRNA precursors from human ESTs and introns.

BACKGROUND: MicroRNAs (miRNAs) function in many physiological processes, and their discovery is beneficial for further studying their physiological functions. However, many of the miRNAs predicted from genomic sequences have not been experimentally validated to be authentic expressed RNA transcripts, thereby decreasing the reliability of miRNA discovery. To overcome this problem, we examined expressed transcripts - ESTs and intronic sequences - to identify novel miRNAs as well as their target genes. RESULTS: To facilitate our approach, we developed our scanning method using criteria based on the features of 207 known human pre-miRNAs to discriminate miRNAs from random sequences. We identified 208 candidate hairpins in human ESTs and human reference gene intronic sequences, 52 of which are known pre-miRNAs. The discovery pipeline performance was further assessed using 130 newly updated pre-miRNA and randomly selected sequences. We achieved sensitivity of 85% (110/130) and overall specificity of 49.7% using this method. Because miRNAs are evolutionarily conserved regulators of gene expression, it is expected that their host genes and target genes should have respective phylogenetic orthologs. Our results confirmed that, in certain mammals, the host genes carrying the same miRNAs are orthologs, as previously reported. Moreover, this observation is also the case for some of the miRNA target genes. CONCLUSION: We have predicted 208 human pre-miRNA candidates and over 10,000 putative human target genes. Using sequence information from ESTs and introns ensures that the predicted pre-miRNA candidates are expressed and the combined expression transcription information from ESTs and introns makes our prediction results more decisive with regard to expressed pre-miRNAs.

3' Untranslated Regions↗

PubMed2006

Methodologies for high-throughput expression profiling of microRNAs.

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression controlling central biological processes. These small, approx 22-nucleotide (nt)-long RNA molecules induce translational suppression when they are imperfectly matched to their target messenger RNA (mRNA) or direct mRNA cleavage when perfectly, or nearly perfectly, matched to their target. Direct roles in developmental processes have been described in a variety of species, and involvement in human diseases, such as cancer and diabetes, has been implied. These studies highlight the need to obtain detailed expression profiles of miRNAs in tissues, during development, and in disease. Their small size and the existence of miRNA families of related sequences pose critical problems in approaching expression analysis of miRNAs, especially using high-throughput approaches. All methodologies presented here address the special requirements for the analysis of miRNA expression using a variety of platforms, including cloning, microarrays, and microbeads. The different variables, as well as the different approaches, used by various laboratories are detailed and general recommendations are provided.

Animals↗

PubMed2005

MicroRNA biogenesis and function in plants.

A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.

Animals↗

PubMed2006

Activation of an oncogenic microRNA cistron by provirus integration.

Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs. Our results demonstrate that retroviral mutagenesis can be a potent tool for miRNA discovery.

Animals↗

PubMed2005

Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila.

In Drosophila melanogaster, Dicer-2/R2D2 and Dicer-1 generate small interfering RNA (siRNA) and microRNA (miRNA), respectively. Here we identify a novel dsRNA-binding protein, which we named R3D1-L, that forms a stable complex with Dicer-1 in vitro and in vivo. While depletion of R3D1-L by RNAi causes accumulation of precursor miRNA (pre-miRNA) in S2 cells, recombinant R3D1-L enhances miRNA production by Dicer-1 in vitro. Furthermore, R3D1 deficiency causes miRNA-generating defect and severe sterility in male and female flies. Therefore, R3D1-L functions in concert with Dicer-1 in miRNA biogenesis and is required for reproductive development in Drosophila.

Adenosine Triphosphate↗

PubMed2005

Antiquity of microRNAs and their targets in land plants.

MicroRNAs (miRNAs) affect the morphology of flowering plants by the posttranscriptional regulation of genes involved in critical developmental events. Understanding the spatial and temporal dynamics of miRNA activity during development is therefore central for understanding miRNA functions. We describe a microarray suitable for detection of plant miRNAs. Profiling of Arabidopsis thaliana miRNAs during normal development extends previous expression analyses, highlighting differential expression of miRNA families within specific organs and tissue types. Comparison of our miRNA expression data with existing mRNA microarray data provided a global intersection of plant miRNA and mRNA expression profiles and revealed that tissues in which a given miRNA is highly expressed are unlikely to also show high expression of the corresponding targets. Expression profiling was also used in a phylogenetic survey to test the depth of plant miRNA conservation. Of the 23 families of miRNAs tested, expression of 11 was detected in a gymnosperm and eight in a fern, directly demonstrating that many plant miRNAs have remained essentially unchanged since before the emergence of flowering plants. We also describe an empirical strategy for detecting miRNA target genes from unsequenced transcriptomes and show that targets in nonflowering plants as deeply branching as ferns and mosses are homologous to the targets in Arabidopsis. Therefore, several individual miRNA regulatory circuits have ancient origins and have remained intact throughout the evolution and diversification of plants.

Arabidopsis↗

PubMed2006

A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses.

We have developed an approach to identify microRNAs (miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes of small size (<2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.

Algorithms↗

PubMed2006

miRBase: the microRNA sequence database.

The miRBase Sequence database is the primary repository for published microRNA (miRNA) sequence and annotation data. miRBase provides a user-friendly web interface for miRNA data, allowing the user to search using key words or sequences, trace links to the primary literature referencing the miRNA discoveries, analyze genomic coordinates and context, and mine relationships between miRNA sequences. miRBase also provides a confidential gene-naming service, assigning official miRNA names to novel genes before their publication. The methods outlined in this chapter describe these functions. miRBase is freely available to all at http://microrna.sanger.ac.uk/.

Animals↗

PubMed2006

MicroRNA expression during chick embryo development.

MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function.

Animals↗