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At least 163 records · Page 9Linked to original sources

PubMed2006

Regulation of pri-microRNA BIC transcription and processing in Burkitt lymphoma.

BIC is a primary microRNA (pri-miR-155) that can be processed to mature miR-155. In this study, we show the crucial involvement of protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) in the regulation of BIC expression upon B-cell receptor triggering. Surprisingly, Northern blot analysis did not reveal any miR-155 expression upon induction of BIC expression in the Burkitt lymphoma-derived Ramos cell line, whereas other microRNAs were clearly detectable. Ectopic expression of BIC in Ramos and HEK293 cells resulted in miR-155 expression in HEK293, but not in Ramos cells, suggesting a specific block of BIC to miR-155 processing in Ramos. In line with the results obtained with Ramos, lack of miR-155 expression after induction of BIC expression was also observed in other Burkitt lymphoma cell lines, indicating a generic and specific blockade in the processing of BIC in Burkitt lymphoma. In contrast, induction of BIC expression in normal tonsillar B cells resulted in very high levels of miR-155 expression and induction of BIC expression in Hodgkin's lymphoma cell lines. It also resulted in elevated levels of miR-155. Our data provide evidence for two levels of regulation for mature miR-155 expression: one at the transcriptional level involving PKC and NF-kappaB, and one at the processing level. Burkitt lymphoma cells not only express low levels of BIC, but also prevent processing of BIC via an, as yet, unknown mechanism.

Blotting, Northern↗

PubMed2005

A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells.

The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that down-regulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Importantly, our shRNA expression system [called PRIME (potent RNA interference using microRNA expression) vectors] allows for the multicistronic cotranscription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations.

Animals↗

PubMed2006

NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-kappaB-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3' UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.

Animals↗

PubMed2006

miR-7b, a microRNA up-regulated in the hypothalamus after chronic hyperosmolar stimulation, inhibits Fos translation.

The transcription factor activator protein 1 (AP-1) is formed through the dimerization of immediate-early genes Fos and Jun family members. Activator protein 1 is known as a pivotal regulator of major biological events such as cell proliferation, differentiation, organogenesis, memory formation, and apoptosis. During a search for microRNAs (miRNAs; small, endogenous, noncoding RNAs that repress gene expression of target mRNAs in animals posttranscriptionally) that are differentially expressed in the mouse paraventricular and supraoptic nuclei after 10 days of drinking 2% saline, one candidate microRNA that is relatively highly expressed, mmu-miR-7b (miR-7b), was studied further because sequence analysis suggested a likely interaction with the 3' untranslated region of Fos mRNA. We show that miR-7b expression inhibits Fos translation in vitro and that it and its host gene are prominently expressed in the PVN and other brain areas, including the suprachiasmatic nucleus. No effect on Fos mRNA levels was observed. Normally, Fos is expressed at low to undetectable levels in cells, but it shows rapid induction and decay after acute stimuli. Various pathways have been identified through which Fos family proteins are degraded; our results indicate a significant additional mechanism by which Fos protein and activity may be regulated.

3' Untranslated Regions↗

PubMed2006

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Animals↗

PubMed2005

Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences.

Drosha is a member of the ribonuclease (RNase) III family that selectively processes RNAs with prominent double-stranded features. Drosha plays a key role in the generation of precursor microRNAs from primary microRNA (pri-miRNA) transcripts in animal cells, yet how Drosha recognizes its RNA substrates remains incompletely understood. Previous studies have indicated that, within the context of a larger pri-miRNA, an approximately 80-nucleotide-long RNA hairpin structure is necessary for processing by Drosha. Here, by performing in vitro Drosha processing reactions with RNA substrates of various sizes and structures, we show that Drosha function also requires single-stranded RNA extensions located outside the pri-miRNA hairpin. The sequence of these RNA extensions was largely unimportant, but a strong secondary structure within the extension or a blunt-ended pri-miRNA hairpin blocked Drosha cleavage. The requirement for single-stranded extensions on the pri-miRNA hairpin substrate for Drosha processing is currently unique among the RNase III enzymes.

Base Sequence↗

PubMed2004

Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences.

MOTIVATION: Most non-coding RNAs are characterized by a specific secondary and tertiary structure that determines their function. Here, we investigate the folding energy of the secondary structure of non-coding RNA sequences, such as microRNA precursors, transfer RNAs and ribosomal RNAs in several eukaryotic taxa. Statistical biases are assessed by a randomization test, in which the predicted minimum free energy of folding is compared with values obtained for structures inferred from randomly shuffling the original sequences. RESULTS: In contrast with transfer RNAs and ribosomal RNAs, the majority of the microRNA sequences clearly exhibit a folding free energy that is considerably lower than that for shuffled sequences, indicating a high tendency in the sequence towards a stable secondary structure. A possible usage of this statistical test in the framework of the detection of genuine miRNA sequences is discussed.

Animals↗

PubMed2005

Human microRNA (miR29b) expression controls the amount of branched chain alpha-ketoacid dehydrogenase complex in a cell.

Branched chain amino acids (BCAAs) play critical roles in cell and tissue functions in addition to being important components of protein structure. The multifunctional roles speak to the need for maintaining tight control of their concentration within cells. As the BCAA cannot be made de novo in mammals, their cellular concentration is a function of dietary intake, endogenous protein turnover and catabolism of the three amino acids. The branched chain alpha-ketoacid dehydrogenase (BCKD) complex commits the BCAA to degradation and thus is vital in controlling their concentration within a cell. In mammals, BCKD activity state depends on the presence of a covalently bound phosphate on one protein component of the complex. Phosphate is added to the protein through the action of the complex-specific kinase and results in BCKD inactivation. Here, we demonstrate that another reaction plays a role in determining the total amount of BCKD present in a cell. The microRNA (miR29b) molecule tested is targeted to the mRNA for the dihydrolipoamide branched chain acyltransferase component of BCKD and prevents translation when bound. This is the first demonstration of the use of a microRNA to exert control on a metabolic pathway of amino acid catabolism in mammals and offers an explanation for the observed differences in the amount of the BCKD complex present in different tissues and under varying nutritional states.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2004

Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins.

Post-transcriptional inhibition of HIV-1 replication can be achieved by RNA interference (RNAi). The cellular expression of short interfering RNA (siRNA) or short hairpin RNA (shRNA) homologous to regions of the HIV-1 genome decreases viral replication by the selective degradation of targeted RNA. Here, we demonstrate that another class of noncoding regulatory RNA, termed microRNA (miRNA), can be used to deliver antiviral RNAi. By incorporating sequences encoding siRNA targeting the HIV-1 transactivator protein tat into a human miR-30 pre-microRNA (pre-miRNA) backbone, we were able to express tat siRNA in cells. The tat siRNA delivered as pre-miRNA precursor was 80% more effective in reducing HIV-1 p24 antigen production than tat siRNA expressed as conventional shRNA. Our results confirm the utility of expressing HIV-1 specific siRNA through a miR-30 precursor stem-loop structure and suggest that this strategy can be used to increase the antiviral potency of RNAi.

Base Sequence↗

PubMed2006

GenomeTrafac: a whole genome resource for the detection of transcription factor binding site clusters associated with conventional and microRNA encoding genes conserved between mouse and human gene orthologs.

Transcriptional cis-regulatory control regions frequently are found within non-coding DNA segments conserved across multi-species gene orthologs. Adopting a systematic gene-centric pipeline approach, we report here the development of a web-accessible database resource--GenomeTraFac (http://genometrafac.cchmc.org)--that allows genome-wide detection and characterization of compositionally similar cis-clusters that occur in gene orthologs between any two genomes for both microRNA genes as well as conventional RNA-encoding genes. Each ortholog gene pair can be scanned to visualize overall conserved sequence regions, and within these, the relative density of conserved cis-element motif clusters form graph peak structures. The results of these analyses can be mined en masse to identify most frequently represented cis-motifs in a list of genes. The system also provides a method for rapid evaluation and visualization of gene model-consistency between orthologs, and facilitates consideration of the potential impact of sequence variation in conserved non-coding regions to impact complex cis-element structures. Using the mouse and human genomes via the NCBI Reference Sequence database and the Sanger Institute miRBase, the system demonstrated the ability to identify validated transcription factor targets within promoter and distal genomic regulatory regions of both conventional and microRNA genes.

Animals↗

PubMed2006

PolymiRTS Database: linking polymorphisms in microRNA target sites with complex traits.

Polymorphism in microRNA Target Site (PolymiRTS) database is a collection of naturally occurring DNA variations in putative microRNA target sites. PolymiRTSs may affect gene expression and cause variations in complex phenotypes. The database integrates sequence polymorphism, phenotype and expression microarray data, and characterizes PolymiRTSs as potential candidates responsible for the quantitative trait locus (QTL) effects. It is a resource for studying PolymiRTSs and their implications in phenotypic variations. PolymiRTS database can be accessed at http://compbio.utmem.edu/miRSNP/.

Animals↗

PubMed2004

MicroRNA-196 inhibits HOXB8 expression in myeloid differentiation of HL60 cells.

MicroRNAs (miRNAs) are endogenous small RNAs (approximately 21-25 nts) that can regulate gene expression at a post-transcriptional level. Recently a large number of miRNAs have been identified from plants and animals. In addition, it has been reported that these small RNAs can participate in cell differentiation of developmental timing. In this study, we show that microRNA-196 (miR-196) inhibits HOXB8 expression in myeloid differentiation of HL 60 cells. MiR-196 has perfect complementarity with target sequence of 3'-UTR in HOXB8 mRNA. Exogenous expressed miR-196 repressed expression of HOXB8 via cleaving mRNAs. Moreover miR-196 enhanced myeloid differentiation of HL60 cells. These results suggest that miR-196 participates in myeloid differentiation of HL60 cells via regulation of HOXB8 expression.

Base Sequence↗

PubMed2004

The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR.

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3'UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3'UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3'UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.

3' Untranslated Regions↗

PubMed2006

Mutations in the microRNA complementarity site of the INCURVATA4 gene perturb meristem function and adaxialize lateral organs in arabidopsis.

Here, we describe how the semidominant, gain-of-function icu4-1 and icu4-2 alleles of the INCURVATA4 (ICU4) gene alter leaf phyllotaxis and cell organization in the root apical meristem, reduce root length, and cause xylem overgrowth in the stem. The ICU4 gene was positionally cloned and found to encode the ATHB15 transcription factor, a class III homeodomain/leucine zipper family member, recently named CORONA. The icu4-1 and icu4-2 alleles bear the same point mutation that affects the microRNA complementarity site of ICU4 and is identical to those of several semidominant alleles of the class III homeodomain/leucine zipper family members PHABULOSA and PHAVOLUTA. The icu4-1 and icu4-2 mutations significantly increase leaf transcript levels of the ICU4 gene. The null hst-1 allele of the HASTY gene, which encodes a nucleocytoplasmic transporter, synergistically interacts with icu4-1, the double mutant displaying partial adaxialization of rosette leaves and carpels. Our results suggest that the ICU4 gene has an adaxializing function and that it is down-regulated by microRNAs that require the HASTY protein for their biogenesis.

Alleles↗

PubMed2004

Managing the genome: microRNAs in Drosophila.

MicroRNAs (miRNAs) represent a growing class of short non-coding RNAs that regulate gene expression by post-transcriptional mechanisms. By binding to target mRNAs via stretches of sequence complementarity, microRNAs inhibit the production of target proteins or induce degradation of mRNAs. Several hundred miRNAs have recently been predicted and cloned from eukaryotic organisms as diverse as plants, invertebrates, and vertebrates. Some miRNAs were shown to be widely conserved across phyla. However, except in a few described cases, rather little is known about their endogenous target genes and the physiological pathways they impinge on. Invertebrate model organisms such as C. elegans and Drosophila have been instrumental to develop methods and to dissect biological roles of miRNAs. In this review, we will focus on recent progress in characterizing miRNAs and steps toward identification of target genes in Drosophila. Many of these recent experiments provide evidence that a systematic target discovery is feasible and that the biology of miRNAs can be functionally explored using forward and reverse genetic tools.

Animals↗

PubMed2005

New human and mouse microRNA genes found by homology search.

Conservation of microRNAs (miRNAs) among species suggests that they bear conserved biological functions. However, sequencing of new miRNAs has not always been accompanied by a search for orthologues in other species. I report herein the results of a systematic search for interspecies orthologues of miRNA precursors, leading to the identification of 35 human and 45 mouse new putative miRNA genes. MicroRNA tracks were written to visualize miRNAs in human and mouse genomes on the UCSC Genome Browser. Based on their localization, miRNA precursors can be excised either from introns or exons of mRNAs. When intronic miRNAs are antisense to the apparent host gene, they appear to originate from ill-characterized antisense transcription units. Exonic miRNAs are, in general, nonprotein-coding, poorly conserved genes in sense orientation. In three cases, the excision of an miRNA from a protein-coding mRNA might lead to the degradation of the rest of the transcript. Moreover, three new examples of miRNAs fully complementary to an mRNA are reported. Among these, miR135a might control the stability and/or translation of an alternative form of the glycerate kinase mRNA by RNA interference. I also discuss the presence of human miRNAs in introns of paralogous genes and in miRNA clusters.

Animals↗

PubMed2006

Developmental and seasonal expression of PtaHB1, a Populus gene encoding a class III HD-Zip protein, is closely associated with secondary growth and inversely correlated with the level of microRNA (miR166).

In contrast to our knowledge of the shoot apical meristem, our understanding of cambium meristem differentiation and maintenance is limited. Class III homeodomain leucine-zipper (HD-Zip) proteins have been shown to play a regulatory role in vascular differentiation. The hybrid aspen (Populus tremulaxPopulus alba) class III HD-Zip transcription factor (PtaHB1) and microRNA 166 (Pta-miR166) family were cloned from hybrid aspen using a combination of in silico and polymerase chain reaction methods. Expression analyses of PtaHB1 and Pta-miR166 were performed by Northern blot analysis. The expression of PtaHB1 was closely associated with wood formation and regulated both developmentally and seasonally, with the highest expression during the active growing season. Also, its expression was inversely correlated with the level of Pta-miR166. Pta-miR166-directed cleavage of PtaHB1 in vivo was confirmed using modified 5'-rapid amplification of cDNA ends (RACE). The expression of Pta-miR166 was much higher in the winter than in the growing seasons, suggesting seasonal and developmental regulation of microRNA in this perennial plant species.

Amino Acid Sequence↗

PubMed2005

A developmental timing microRNA and its target regulate life span in C. elegans.

The microRNA lin-4 and its target, the putative transcription factor lin-14, control the timing of larval development in Caenorhabditis elegans. Here, we report that lin-4 and lin-14 also regulate life span in the adult. Reducing the activity of lin-4 shortened life span and accelerated tissue aging, whereas overexpressing lin-4 or reducing the activity of lin-14 extended life span. Lifespan extension conferred by a reduction in lin-14 was dependent on the DAF-16 and HSF-1 transcription factors, suggesting that the lin-4-lin-14 pair affects life span through the insulin/insulin-like growth factor-1 pathway. This work reveals a role for microRNAs and developmental timing genes in life-span regulation.

Aging↗