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PubMed2006

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed.

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

Cell Line↗

PubMed2004

Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning.

MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the approximately 22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polymerase chain reaction. cDNAs are cloned in lambda or plasmid vectors, and the sequences are compared to annotated genomic sequence databases, and analyzed by RNA folding programs to distinguish miRNA sequences from other small RNAs of similar size. Northern blot hybridization is used to confirm the expression of small RNAs in vivo.

3' Untranslated Regions↗

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

The microRNA: overview of the RNA gene that modulates gene functions.

MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both messenger RNA (mRNA) degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA. Different names have been used to describe various types of miRNA. During evolution, RNA retroviruses or transgenes invaded the eukaryotic genome and inserted itself in the noncoding regions of DNA, conceivably acting as transposon-like jumping genes, providing defense from viral invasion and fine-tuning of gene expression as a secondary level of gene modulation in eukaryotes. When a transposon is inserted in the intron, it becomes an intronic miRNA, taking advantage of the protein synthesis machinery, i.e., mRNA transcription and splicing, as a means for processing and maturation. Recently, miRNAs have been found to play an important, but not life-threatening, role in embryonic development. They might play a pivotal role in diverse biological systems in various organisms, facilitating a quick response and accurate plotting of body physiology and structures. Based on these unique properties, manufactured intronic miRNAs have been developed for in vitro evaluation of gene function, in vivo gene therapy, and generation of transgenic animal models. The biogenesis and identification of miRNAs, potential applications, and future directions for research are presented in this chapter, hopefully providing a guideline for further miRNA and gene function studies.

Animals↗

PubMed2006

Prediction of human microRNA targets.

MicroRNAs (miRNAs) are small, nonprotein-coding RNAs that regulate gene expression. Although hundreds of human miRNA genes have been discovered, the functions of most of these are unknown. Computational predictions indicate that miRNAs, which account for at least 1% of human protein-coding genes, regulate protein production for thousands of or possibly all of human genes. We discuss the functions of mammalian miRNAs and the experimental and computational methods used to detect and predict human miRNA target genes. Anticipating their impact on genome-wide discovery of miRNA targets, we describe the various computational tools and web-based resources available to predict miRNA targets.

3' Untranslated Regions↗

PubMed2006

Complications in mammalian microRNA target prediction.

In this chapter, we review evidence that at least three different types of microRNA (miRNA)-messenger RNA (mRNA) target interactions exist in mammals: short seeds, long seeds, and "perfect" hits (allowing G:U matches). Because new types of miRNAs are still being discovered, this list may not yet be complete.

3' Untranslated Regions↗

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

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↗

PubMed2006

In situ hybridization as a tool to study the role of microRNAs in plant development.

MicroRNAs (miRNAs) have a vital role in the generation of plant forms through post-transcriptional regulation of the accumulation of developmental regulators. Analysis of their roles requires detailed knowledge of their expression patterns. We describe an in situ hybridization technique we have used to study the patterns of miRNA accumulation in Arabidopsis and in maize.

Gene Expression Regulation, Plant↗

PubMed2006

Cloning microRNAs from mammalian tissues.

MicroRNAs (miRNAs) are ubiquitous regulators of gene expression in plants and animals. Their distinctive structure, as very short RNAs with a 5'-phosphate and 3'-hydroxyl group, has enabled the development of protocols to clone miRNAs. After enrichment of these small molecules by size, serial ligation of adapter oligonucleotides to each terminus allows amplification using reverse transcription (RT)-polymerase chain reaction (PCR). Plasmid cloning of multiple miRNA sequences and subsequent DNA sequence analysis enable both bioinformatic characterization of the various miRNAs and experimental validation of their accumulation in cells.

Animals↗

PubMed2006

Methods for analyzing microRNA expression and function during hematopoietic lineage differentiation.

MicroRNAs (miRNAs), an abundant class of approx 22-nucleotide (nt) small RNAs that control gene expression at the posttranscriptional level, may play important roles during normal hematopoiesis and leukemogenesis. This chapter focuses on the methods and strategies for dissecting miRNA function during hematopoietic lineage differentiation. We describe a modified miRNA cloning method and expression analysis approach for determining miRNA expression during hematopoietic lineage differentiation. We illustrate a retroviral vector and a general strategy for the ectopic expression of miRNAs in hemato-poietic stem/progenitor cells. We discuss in vitro and in vivo functional assays that can be used to examine the roles of miRNAs during hematopoietic lineage differentiation. The methods and principles described here should also be applicable to study the roles of miRNAs in the differentiation and function of nonhematopoietic cell types.

Animals↗

PubMed2006

Identifying microRNA regulators of cell death in Drosophila.

Animal genomes contain on the order of at least hundreds of microRNAs (miRNAs). Although most remain uncharacterized, it is already clear that miRNAs regulate many biological processes. A number of Drosophila miRNAs have been identified as likely cell death regulators, but functions for most have simply not been explored. Here we describe a protocol for identifying miRNAs that can act as cell death regulators. We also describe a simple protocol for testing roles for mRNAs identified as candidate miRNA targets using computational or other approaches.

Animals↗

PubMed2006

MicroRNAs in human immunodeficiency virus-1 infection.

Initially reported for Caenorhabditis elegans, microRNA (miRNA) has been shown to regulate gene expression in plants, flies, and mammals . Here, we report on our approaches to investigate the role of miRNAs in human immunodeficiency virus (HIV)-1 infection. Using computer-directed foldings, we first identify potential sequences in HIV-1 that putatively encode miRNAs. Subsequently, we use Northern blotting of RNAs isolated from HIV-infected cells to confirm expression of predicted miRNA sequences. Finally, we use a scanning algorithm to search 3' untranslated regions (UTRs) of human messenger RNAs (mRNAs) in the attempt to predict potential sites targeted by HIV-1 miRNAs.

Algorithms↗

PubMed2006

Cloning and detection of HIV-1-encoded microRNA.

MicroRNAs (miRNAs) are 21-to 25-nucleotides (nt) long and interact with messenger RNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi). We have shown that HIV-1 nef double-stranded RNA from AIDS patients who are long-term nonprogressors, inhibits HIV-1 transcription; and that nef-derived miRNA, miR-N367, is produced in human T-cells persistently infected with HIV-1. The miR-N367 can block HIV-1 Nef expression and long terminal repeat (LTR) transcription, suggesting that miR-N367 might suppress both Nef function and HIV-1 transcription through the RNAi pathway. Protocols are presented here for cloning HIV-1-encoded miRNA and confirming miRNA expression by Northern blot hybridization.

Blotting, Northern↗

PubMed2006

Identification of messenger RNAs and microRNAs associated with fragile X mental retardation protein.

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of the Fragile X mental retardation protein (FMRP). FMRP, which may regulate translation in neurons, not only associates with specific messenger RNAs (mRNAs) and with microRNAs (miRNAs), but also associates with the components of the miRNA pathway, including the Dicer and Argonaute proteins. It has been proposed that FMRP regulates the translation of its mRNA targets through miRNAs. In this chapter, we describe the protocol to identify the mRNAs and miRNAs associated with FMRP in vivo. The same method could also be applied to other RNA-binding proteins interacting with specific mRNAs or miRNAs.

Animals↗

PubMed2006

In vitro precursor microRNA processing assays using Drosophila Schneider-2 cell lysates.

Recent studies have shown that the microRNA (miRNA) pathway is an evolutionarily conserved endogenous pathway that is important for normal development. Mature miRNAs are excised from precursors in a stepwise process and subsequently incorporated into an RNA-induced silencing complex (RISC), which mediates either cleavage of the target messenger RNA (mRNA) or translational repression, depending on the complementarity between the miRNA and its target mRNA. In this chapter, we describe in vitro precursor (pre)-miRNA processing assays using Drosophila Schneider-2 (S2) cell lysates and immunopurified materials.

Animals↗

PubMed2006

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest clinical and circumstantial malfunction. Type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonproteincoding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Animals↗

PubMed2006

Isolation and identification of gene-specific microRNAs.

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Animals↗