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PubMed2006

Transgene-like animal models using intronic microRNAs.

Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude of a human body for etiological and pathological research of diseases. All pharmaceutically developed drugs must be proven safe and effective in animals before approval by the Food and Drug Administration to be used in clinical trials. To this end, the transgenic animal models of human diseases serve as a front line for drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA) research. miRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts that contain either complete or partial complementarity to the miRNAs, are useful for the design and development of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse, and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerase (Pol)-II, coexpressed with the encoding gene transcript, and excised out of the encoding gene transcript by natural RNA splicing and processing mechanisms. In conjunction with retroviral transfection systems, the hairpin-like pre-miRNA construct was further inserted into the intron of a cellular gene for tissue-specific expression regulated by the gene promoter. Because the retroviral vectors were randomly integrated into the genome of its host cell, the most effective transgenic animal can be selected and propagated to be a stable transgenic line for future research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA-expressing system described previously, which has been proven to be useful for both miRNA research and in vivo evaluation of miRNA-associated target genes.

Animals↗

PubMed2006

MicroRNA biogenesis: isolation and characterization of the microprocessor complex.

The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that have been implicated in controlling diverse pathways in a variety of organisms (1, 2). For posttranscriptional gene silencing, one strand of the miRNA is used to guide components of the RNA interference machinery, including Argonaute 2, to messenger RNAs (mRNAs) with complementary sequences (3, 4). Thus, targeted mRNAs are either cleaved by the endonuclease Argonaute 2 (5, 6), or protein synthesis is blocked by an as yet uncharacterized mechanism (7, 8). Genes encoding miRNAs are transcribed as long primary miRNAs (pri-miRNAs) that are sequentially processed by components of the nucleus and cytoplasm to yield a mature, approx 22-nucleotide (nt)-long miRNA (9). Two members of the ribonuclease (RNase) III endonuclease protein family, Drosha and Dicer, have been implicated in this two-step processing (10-13). To further our understanding of miRNA biogenesis and function it will be essential to identify the protein complexes involved. We were interested in defining the proteins required for the initial nuclear processing of pri-miRNAs to the approx 60- to 70-nt stem-loop intermediates known as precursor miRNAs (pre-miRNAs) (9, 10). This led to our identification of a protein complex we termed Microprocessor, which is necessary and sufficient for processing pri-miRNA to premiRNAs (14). The Microprocessor complex comprises Drosha and the double-stranded RNAbinding protein DiGeorge syndrome critical region 8 gene (DGCR8), which is deleted in DiGeorge syndrome (15, 16). In this chapter, we detail the methods used for the biochemical isolation and identification of the Microprocessor complex from human cells. We include a protocol for the in vitro analysis of pri-miRNA processing activity of the purified Microprocessor complex.

Cell Line↗

PubMed2006

Evolution of microRNAs.

MicroRNAs (miRNAs) form a large class of small regulatory RNAs in eukaryotes. Although they share a common processing pathway and certain structural features, in general, there is no detectable sequence similarity among miRNAs from a given organism. On the other hand, many miRNAs are members of a family of a few, often very similar, paralogs. It is, thus, of interest to trace the evolutionary history of individual miRNAs, to identify the timing of gene duplications, and to study relationships between the histories of different miRNA families. Some miRNAs are transcribed from polycistronic primary transcripts. In these cases, we will study the evolution of entire clusters.

Base Pairing↗

PubMed2006

MicroRNA protocols. Perspectives.

The discoveries of microRNAs (miRNAs), a class of noncoding RNAs that can regulate gene expression by translational repression, have opened a new avenue on gene modulation in mammalian cells. Conceivably, this highly effective method of modulation of gene expression would be exploited for numerous prospectives, including human, therapeutics. This chapter offers some perspective, with emphasis on areas that can be further developed.

Animals↗

PubMed2006

Recognition and cleavage of primary microRNA transcripts.

MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary miRNAs (pri-miRNAs), the much longer precursors of mature miRNAs, are processed in human cells, as well as in vitro.

Humans↗

PubMed2006

MicroRNAs and messenger RNA turnover.

Although initially believed to act exclusively as translational repressors, microRNAs (miRNAs) are now known to target complementary messenger RNA (mRNA) transcripts for either translational repression or cleavage via the RNA-induced silencing complex (RISC) ([1], reviewed in ref. 2). The current model postulates that mature miRNAs are incorporated into the RISC, bind target mRNAs based on complementarity, and guide cleavage of mRNA targets with perfect or nearly perfect complementarity and translational repression of targets with lower complementarity (2). The translational repression mechanism of miRNA-mediated gene regulation, which is common in animals but also exists in plants, is not well understood mechanistically. Conversely, miRNA-directed mRNA cleavage by RISC is common in plants, but also occurs in animals (3). This chapter focuses on the mRNA cleavage by miRNA-programmed RISC, and, specifically, on characterizing the products of such cleavage.

Animals↗

PubMed2006

Prediction of microRNA targets.

I describe the use of RNAhybrid, a program that predicts multiple potential binding sites of microRNAs (miRNAs) in large target RNAs. The core algorithm finds the energetically most favorable hybridization sites of a miRNA in a large potential target RNA. Intramolecular hybridizations, i.e., base pairings between target nucleotides or between miRNA nucleotides are, not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Starting from the observation that the binding energies are results from an optimization procedure, we can model them as following an extreme value distribution. From this, we can calculate the statistical significance of individual binding sites, of multiple binding sites in a single target sequence, and of binding sites in comparative analyses of orthologous sequences across species. The latter involves the calculation of the effective number of orthologous sequences, which can be considerably smaller than the actual number, reflecting the statistical dependence of evolutionarily related sequences.

Algorithms↗

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↗

PubMed2005

Plant microRNAs and development.

MicroRNAs (miRNAs) act as negative regulators of gene expression in eukaryotes, a discovery that has opened an expanding field of biological research. Plant miRNAs are known to repress gene expression posttranscriptionally, mainly by guiding cleavage but also by attenuating the translation of target transcripts. In addition, it has been shown that plant miRNAs can also act at the transcriptional level by directing the methylation of target chromosomal loci. Genetic and biochemical approaches are quickly broadening our knowledge of the biogenesis and function of plant miRNAs. Computational approaches have uncovered an unexpectedly large number of miRNAs and their targets in plants. The targets of plant miRNAs often belong to families of transcription factors involved in the control of developmental processes. We review the status of research in this dynamic field, summarizing recent advances in our understanding of the biogenesis and mechanism of action of plant miRNAs, as well as in the developmental processes they regulate.

Base Sequence↗

PubMed2005

microRNA-guided posttranscriptional gene regulation.

microRNAs (miRNAs) form an evolutionarily conserved and highly abundant class of non-coding RNAs that are 21-24 nucleotides (nt) in length. They are processed from double-stranded (ds) RNA precursors and sequence-specifically guide posttranscriptional gene silencing. The processing steps are facilitated by members of the RNAse III enzyme family, whereas gene silencing events are mediated by members of the highly conserved Argonaute (Ago) protein family. Initially discovered in Caenorhabditis elegans, in which they are essential for proper developmental timing, hundreds of miRNAs have been discovered to date in a variety of different organisms, including plants, flies and mammals. Expression profiling approaches demonstrated that miRNAs are specifically expressed not only during embryonic development, but also during cell differentiation and other cellular events such as hormonal signaling. Although miRNAs have been the object of extensive research in recent years, very little is known about their target mRNAs. Their identification along with a comprehensive description of the miRNA/target-mRNA interaction network will add a new level to our knowledge of gene regulation and will also provide new insights into the biology of so far poorly understood diseases, including various forms of cancer.

Animals↗

PubMed2006

[MicroRNAs: novel class of gene regulators involved in endocrine function and cancer].

MicroRNAs (miRNAs) represent a novel class of endogenous approximately 22-nucleotide RNAs that negatively regulate gene expression by inhibiting translation of target RNAs. Discovered just over a decade ago in Caenorhabditis elegans, miRNAs are now recognized as one of the major regulatory gene families in plants and animals. In the human genome, 462 miRNA genes have been discovered and the estimated number of miRNAs is as high as 1000. Bioinformatics analysis indicated that a unique miRNA acts on several mRNA, influencing multiple signaling pathways concomitantly, thus presenting enormous regulatory potential. Although the biology of miRNAs is not well understood, recent evidences have linked these molecules to diverse biological processes. Moreover, aberrant expression of miRNAs has been associated to human disease, including that related to the endocrine system and cancer.

Adipocytes↗

PubMed2006

Computational prediction of microRNA genes in silkworm genome.

MicroRNAs (miRNAs) constitute a novel, extensive class of small RNAs (approximately 21 nucleotides), and play important gene-regulation roles during growth and development in various organisms. Here we conducted a homology search to identify homologs of previously validated miRNAs from silkworm genome. We identified 24 potential miRNA genes, and gave each of them a name according to the common criteria. Interestingly, we found that a great number of newly identified miRNAs were conserved in silkworm and Drosophila, and family alignment revealed that miRNA families might possess single nucleotide polymorphisms. miRNA gene clusters and possible functions of complement miRNA pairs are discussed.

Animals↗

PubMed2005

Specific microRNAs modulate embryonic stem cell-derived neurogenesis.

MicroRNAs (miRNAs) are recently discovered small non-coding transcripts with a broad spectrum of functions described mostly in invertebrates. As post-transcriptional regulators of gene expression, miRNAs trigger target mRNA degradation or translational repression. Although hundreds of miRNAs have been cloned from a variety of mammalian tissues and cells and multiple mRNA targets have been predicted, little is known about their functions. So far, a role of miRNA has only been described in hematopoietic, adipocytic, and muscle differentiation; regulation of insulin secretion; and potentially regulation of cancer growth. Here, we describe miRNA expression profiling in mouse embryonic stem (ES) cell- derived neurogenesis in vitro and show that a number of miRNAs are simultaneously co-induced during differentiation of neural progenitor cells to neurons and astrocytes. There was a clear correlation between miRNA expression profiles in ES cell-derived neurogenesis in vitro and in embryonal neurogenesis in vivo. Using both gain-of-function and loss-of-function approaches, we demonstrate that brain-specific miR-124a and miR-9 molecules affect neural lineage differentiation in the ES cell-derived cultures. In addition, we provide evidence that signal transducer and activator of transcription (STAT) 3, a member of the STAT family pathway, is involved in the function of these miRNAs. We conclude that distinct miRNAs play a functional role in the determination of neural fates in ES cell differentiation.

Animals↗

PubMed2006

MicroRNA deregulation in human thyroid papillary carcinomas.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in a wide range of basic processes such as cell proliferation, development, apoptosis and stress response. It has recently been found that they are also abnormally expressed in many types of human cancer. We analyzed the genome-wide miRNA expression profile in human thyroid papillary carcinomas (PTCs) using a microarray (miRNACHIP microarray) containing hundreds of human precursor and mature miRNA oligonucleotide probes. Using this approach, we found an aberrant miRNA expression profile that clearly differentiates PTCs from normal thyroid tissues. In particular, a significant increase in miRNA (miR)-221, -222 and -181b was detected in PTCs in comparison with normal thyroid tissue. These results were further confirmed by northern blot and quantitative RT-PCR analyses. Moreover, RT-PCR revealed miR-221, -222 and -181b overexpression in fine needle aspiration biopsies corresponding to thyroid nodules, which were eventually diagnosed as papillary carcinomas after surgery. Finally, miR-221, -222 and -181b overexpression was also demonstrated in transformed rat thyroid cell lines and in mouse models of thyroid carcinogenesis. Functional studies, performed by blocking miR-221 function and by overexpressing miR-221 in human PTC-derived cell lines, suggest a critical role of miR-221 overexpression in thyroid carcinogenesis. In conclusion, these data, taken together, indicate an miRNA signature associated with PTCs, and suggest miRNA deregulation as an important event in thyroid cell transformation.

Animals↗

PubMed2005

MicroRNAs and endocrine biology.

microRNAs (miRNAs) are highly conserved, non-coding RNAs that powerfully regulate gene expression at the post-transcriptional level. These fascinating molecules play essential roles in many biological processes in mammals, including insulin secretion, B-cell development, and adipocyte differentiation. This review provides a general background regarding current knowledge about miRNA biogenesis and the potential contributions of these RNAs to endocrine function.

Adipocytes↗

PubMed2005

Facile means for quantifying microRNA expression by real-time PCR.

MicroRNAs (miRNAs) are 20-24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having asfew as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.

Arabidopsis↗

PubMed2006

Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid.

The detection of microRNAs (miRNAs) at single-cell resolution is important for studying the role of these posttranscriptional regulators. Here, we use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid, injected into zebrafish blastomeres or electroporated into defined tissues of mouse embryos in utero or ex utero, to monitor the dynamics of specific miRNAs in individual live cells. This approach reveals, for example, that in the developing mouse central nervous system, miR-124a is expressed not only in postmitotic neurons but also in neuronal progenitor cells. Collectively, our results demonstrate that acute administration of DFRS plasmids offers an alternative to previous in situ hybridization and transgenic approaches and allows the monitoring of miRNA appearance and disappearance in defined cell lineages during vertebrate development.

Animals↗

PubMed2004

Profiling microRNA expression using sensitive cDNA probes and filter arrays.

MicroRNAs (miRNAs) are small noncoding RNAs (approximately 22 nucleotides) that have recently emerged as important regulators of gene expression in both plants and animals. With few exceptions, however, the target genes and the expression levels of most miRNAs are unknown. Here we show that direct random-primed cDNA synthesis on either chemically synthesized small RNAs (21-22 nucleotides) or gel-purified mature miRNAs from human cells can produce specific and sensitive full-length cDNA probes. Using oligonucleotide filter arrays, we demonstrate that the internally labeled cDNA probes are sensitive for detecting differential miRNA expression between untreated and O-tetradecanoylphorbol-13-acetate (TPA)-treated HL60 cells. The present study should facilitate a high-throughput analysis of miRNA expression between samples.

DNA Probes↗