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At least 919 records · Page 51Linked to original sources
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↗
PubMed2003

Translation repression by antisense sequences.

Antisense sequences that hybridize to messenger RNA can inhibit target gene expression in a variety of ways. The best-known antisense mechanisms trigger messenger RNA (mRNA) degradation; however, translation repression by ribosome interference is a common natural antisense mechanism. In bacteria, there are fascinating examples of cis-encoded and trans-encoded antisense sequences that reversibly repress translation. In eukaryotes, microRNAs that bind 3'UTR target sequences also repress translation, although the mechanism is unclear. An important feature of translation repression is that the mRNA can remain intact during periods of repression, and rapid expression switching can occur in response to cellular and environmental signals. Recent genome analyses indicate many new short noncoding RNAs with predicted antisense activities. Therefore, translation repression by antisense sequences is likely to be a common and important form of posttranscriptional gene control, and such natural mechanisms provide a basis for the development of synthetic antisense gene control in research and drug development.

Animals↗
PubMed2026

Design, expression, purification, and application of novel recombinant miR-491 molecules to define the biogenesis and function of miR-491-3p versus -5p in posttranscriptional regulation of UDP-glucuronosyltransferase 1A1.

Interindividual variations in drug metabolism involve various factors, including posttranscriptional gene regulation mechanisms controlled by microRNAs (miRNAs or miRs) derived from the genome. The aim of this study was to use RNA bioengineering technology to produce novel recombinant human miR-491-5p, miR-491-3p, and pre-miR-491 molecules, namely BioRNA/miR-491-5p, BioRNA/miR-491-3p, and BioRNA/pre-miR-491, respectively, and define their functional difference in regulating UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and drug-metabolizing capacity. All 6 BioRNAs were heterologously overexpressed in Escherichia coli (>30% of total RNA) and isolated by fast protein liquid chromatography to high purity (>97%). As BioRNA/pre-miR-491 agents were processed to both 5p and 3p strands in Hep3B and HepG2 cells, BioRNA/miR-491-5p and -3p were selectively processed to 5p and 3p, respectively, and each accumulated to greater levels. Immunoblotting and immunofluorescence studies demonstrated the efficacy of BioRNA/miR-491-3p to suppress UGT1A1 protein levels in Hep3B and HepG2 cells, localized on the endoplasmic reticulum, exhibiting monomeric (∼55 kDa) and oligomeric (∼150 kDa) bands under different conditions, whereas BioRNA/pre-miR-491 and miR-491-5p had no effects. Using a fluorescent substrate, N-butyl-4-(4-hydroxyphenyl)-1,8-naphthalimide, lower UGT1A1 drug-metabolizing capacities were found in cells treated with BioRNA/miR-491-3p. In addition, liquid chromatography-tandem mass spectrometry analysis revealed a 45% reduction of estradiol 3-glucuronidation activity by BioRNA/miR-491-3p in Hep3B cells, whereas formation of estradiol 17-glucuronidation mediated by other UGTs was unchanged. Together, these results underline the role of miR-491-3p in regulating UGT1A1 and its impact on cellular drug-metabolizing capacity while demonstrating the applications of recombinant miRNA agents to delineating the importance of posttranscriptional gene regulation in drug metabolism. SIGNIFICANT STATEMENT: Research on posttranscriptional gene regulation mainly uses miRNA mimics chemically synthesized in vitro. This study successfully produced 6 novel recombinant miR-491 molecules through in vivo fermentation with transfer RNA scaffold and transfer RNA-fused pre-miRNA carrier-based technologies, which were further utilized to delineate the biogenesis and function of miR-491-3p versus -5p in modulating UDP-glucuronosyltransferase 1A1 protein levels and drug-metabolizing capacity. The findings demonstrate the role of miR-491-3p in regulating UDP-glucuronosyltransferase 1A1 and value of recombinant miRNA agents for studying drug metabolism.

Humans↗
PubMed2006

Frequent loss of Dab2 protein and infrequent promoter hypermethylation in breast cancer.

Disabled-2 (Dab2), a putative tumor suppressor protein, is lost in 80-90% ovarian tumors and ovarian/breast cancer cell lines. The clinical significance of Dab2 protein in breast cancer remains yet unknown. Immunohistochemical analysis of Dab2 protein showed no detectable expression in 67/91 (74%) breast tumors, while all 10 normal tissues showed presence of Dab2 protein. We hypothesized that epigenetic silencing of Dab2 may account for loss of protein in breast cancer. Methylation of Dab2 exon 1, a putative promoter, was analyzed in six breast cancer cell lines and in 54 primary breast tumors by methylation specific PCR. Methylation was observed in MDA-MB-231 and MDA-MB-157 cells and in 6 of 54 (11%) primary breast tumors that also showed loss of Dab2 protein. Expression of Dab2 transcripts was detected in all cell lines except MDA-MB-157. However, none of these six cell lines showed detectable levels of Dab2 protein by western blotting, while non-malignant mammary epithelial cell line MCF 10A showed Dab2 protein expression. To our knowledge this is the first report showing low frequency of Dab2 (putative) promoter methylation (11%) in primary breast tumors. Frequent loss of Dab2 protein (74%) suggest that hypermethylation of Dab2 promoter may only be one of the mechanisms accounting for its loss in breast cancer. Further, in silico analysis of Dab2 3'-UTR revealed existence of miRNA complimentary to this region of the gene, suggesting microRNA mediated targeting of Dab2 mRNA might account for loss of the protein in breast cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Genomic approaches to drug discovery.

Considerable progress has been made in exploiting the enormous amount of genomic and genetic information for the identification of potential targets for drug discovery and development. New tools that incorporate pathway information have been developed for gene expression data mining to reflect differences in pathways in normal and disease states. In addition, forward and reverse genetics used in a high-throughput mode with full-length cDNA and RNAi libraries enable the direct identification of components of signaling pathways. The discovery of the regulatory function of microRNAs highlights the importance of continuing the investigation of the genome with sophisticated tools. Furthermore, epigenetic information including DNA methylation and histone modifications that mediate important biological processes add to the possibilities to identify novel drug targets and patient populations that will benefit from new therapies.

Animals↗
PubMed2006

Gene-expressed RNA as a therapeutic: issues to consider, using ribozymes and small hairpin RNA as specific examples.

In recent years there has been a greater appreciation of both the role of RNA in intracellular gene regulation and the potential to use RNA in therapeutic modalities. In the latter case, RNA can be used as a therapeutic target or a drug. The chapters in this volume cover the varied and potent actions of RNA as antisense, ribozymes, aptamers, microRNA and small hairpin RNA in gene regulation, as well as their use as potential therapeutics for metabolic and infectious diseases. Our group has been involved in the development of anti-HIV gene expression constructs to treat HIV. In this chapter, we address the relevant scientific and some of the commercial issues in the use of RNA as a therapeutic. Specifically, the chapter discusses delivery, expression, potency, toxicity and commercial development using, as examples, hammerhead ribozymes and small hairpin RNA.

Animals↗
PubMed2025

Expression regulation network in papillae of sea cucumbers: Whole-transcriptome and DNA methylation datasets.

To elucidate the expression regulation network of papilla size of sea cucumbers (Apostichopus japonicus), the whole-transcriptome and DNA methylome datasets of different sizes of papillae in sea cucumbers were generated. Average clean bases of whole-transcriptome (16.35 G) and DNA methylome (28.92 G) were obtained using RNA sequencing and whole-genome bisulfite sequencing techniques. A total of 3,188 ceRNA networks were also identified including 3,081 long non-coding RNAs (lncRNA)/microRNAs (miRNA)/mRNA networks and 107 circular RNA (circRNA)/miRNA/mRNA networks. Methylome data indicate that there were 3,307 and 3,776 differentially methylated regions (DMRs) with high-level methylation as well as 3,125 and 3,016 DMRs with low-level methylation in big papillae compared to small papillae. The identified DMRs were mainly distributed in introns, promotors, or exons. The whole-transcriptome and DNA methylome datasets generated from this study not only established a robust theoretical foundation (especially from the epigenetic aspect) for elucidating expression regulation network determining papilla size in sea cucumbers but also can be a valuable resource of biomarker mining for papilla appearance-based selective breeding in sea cucumbers.

DNA Methylation↗
PubMed2005

RDR6 has a broad-spectrum but temperature-dependent antiviral defense role in Nicotiana benthamiana.

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

Arabidopsis↗
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↗
PubMed2004

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes.

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.

Animals↗
PubMed2006

New porcine microRNA genes found by homology search.

MicroRNAs (miRNAs) repress target genes at the post-transcriptional level and play important roles in development and cell lineage decisions. In vertebrates, however, both the targets of miRNAs and their expression profiles during development are poorly understood. Thus far, 326 human miRNAs, 249 mouse miRNAs, and 195 rat miRNAs have been identified. Even though the pig is a promising animal model in regenerative biology, as well as in livestock production, only 54 pig miRNAs have been identified. Here, we report the identification of 58 new pig miRNAs. We conducted a homology search using either human or mouse miRNAs genes queried against the pig genome. The new pig miRNAs consist of 23 miRNAs showing homology to mouse, 21 showing homology to human, and 16 showing homology to both human and mouse. The expression profile of 6 miRNAs were analyzed by Northern blot analysis isolated from porcine tissues: ovary, heart, kidney, liver, and pancreas.

Animals↗
PubMed2006

MicroRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation.

MicroRNAs (miRNAs) are small non-coding RNAs emerging as important post-transcriptional gene regulators. In this study, we examined the role of miR-1, an miRNA specifically expressed in cardiac and skeletal muscle tissue, on the myogenic, osteoblastic, and adipogenic differentiation of C2C12 cells. Upon induction of myogenic differentiation, miR-1 was robustly expressed. Retrovirus-mediated overexpression of miR-1 markedly enhanced expression of muscle creatine kinase, sarcomeric myosin, and alpha-actinin, while the effects on myogenin and MyoD expression were modest. Formation of myotubes was significantly augmented in miR-1-overexpressing cells, indicating miR-1 expression enhanced not only myogenic differentiation but also maturation into myotubes. In contrast, osteoblastic and adipogenic differentiation was not affected by forced expression of miR-1. Thus, the muscle-specific miRNA, miR-1, plays important roles in controlling myogenic differentiation and maturation in lineage-committed cells, rather than functioning in fate determination.

3T3-L1 Cells↗
PubMed2005

Core transcriptional regulatory circuitry in human embryonic stem cells.

The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.

Animals↗
PubMed2006

Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

Activin Receptors, Type I↗
PubMed2003

siRNAs can function as miRNAs.

With the discovery of RNA interference (RNAi) and related phenomena, new regulatory roles attributed to RNA continue to emerge. Here we show, in mammalian tissue culture, that a short interfering RNA (siRNA) can repress expression of a target mRNA with partially complementary binding sites in its 3' UTR, much like the demonstrated function of endogenously encoded microRNAs (miRNAs). The mechanism for this repression is cooperative, distinct from the catalytic mechanism of mRNA cleavage by siRNAs. The use of siRNAs to study translational repression holds promise for dissecting the sequence and structural determinants and general mechanism of gene repression by miRNAs.

3' Untranslated Regions↗
PubMed2005

La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA.

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.

Animals↗
PubMed2005

Silencing of microRNAs in vivo with 'antagomirs'.

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

3' Untranslated Regions↗