Results 1 - 10 of 255

Real-time quantification of microRNAs by stem-loop RT-PCR

by Caifu Chen, Dana A. Ridzon, Adam J. Broomer, Zhaohui Zhou, Danny H. Lee, Julie T. Nguyen, Maura Barbisin, Nan Lan Xu, Vikram R. Mahuvakar, Mark R. Andersen, Kai Qin Lao, Kenneth J. Livak, Karl J. Guegler - Nucleic Acids Res , 2005
"... A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related mi ..."
Abstract - Cited by 394 (8 self) - Add to MetaCart
A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes

by Milo J. Aukerman, Hajime Sakai, The Plant Cell - Plant Cell , 2003
"... MicroRNAs (miRNAs) are �21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating te ..."
Abstract - Cited by 269 (0 self) - Add to MetaCart
MicroRNAs (miRNAs) are �21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.
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RNA polymerase III transcribes human microRNAs.

by Glen M Borchert , William Lanier , Beverly L Davidson - Nature Struct. Mol. Biol. , 2006
"... Prior work demonstrates that mammalian microRNA (miRNA or miR) expression requires RNA polymerase II (Pol II). However, the transcriptional requirements of many miRNAs remain untested. Our genomic analysis of miRNAs in the human chromosome 19 miRNA cluster (C19MC) revealed that they are intersperse ..."
Abstract - Cited by 201 (3 self) - Add to MetaCart
Prior work demonstrates that mammalian microRNA (miRNA or miR) expression requires RNA polymerase II (Pol II). However, the transcriptional requirements of many miRNAs remain untested. Our genomic analysis of miRNAs in the human chromosome 19 miRNA cluster (C19MC) revealed that they are interspersed among Alu repeats. Because Alu transcription occurs through RNA Pol III recruitment, and we found that Alu elements upstream of C19MC miRNAs retain sequences important for Pol III activity, we tested the promoter requirements of C19MC miRNAs. Chromatin immunoprecipitation and cell-free transcription assays showed that Pol III, but not Pol II, is associated with miRNA genomic sequence and sufficient for transcription. Moreover, the mature miRNA sequences of approximately 50 additional human miRNAs lie within Alu and other known repetitive elements. These findings extend the current view of miRNA origins and the transcriptional machinery driving their expression. Over 450 miRNAs have been described in the human genome 1 . miRNAs are important in human development, oncogenesis and immunity, and they have drawn renewed attention to the small noncoding elements of the transcriptome. miRNAs are short (17-25 base pairs (bp)) noncoding RNAs that guide cellular machinery to specific messenger RNAs 2,3 to control expression. Initial miRNA transcripts can be several thousand base pairs in length, and they are processed to produce B70-bp stem-loops (pre-miRNAs) before nuclear export 4 . Upon entering the cytoplasm, the RNA-induced silencing complex (RISC) cleaves and denatures pre-miRNAs to produce the functionally mature, single-stranded miRNAs 4 . Through complementary base pairing to specific protein-coding mRNA transcripts, miRNAs direct mRNA silencing by a variety of mechanisms, including message degradation 5 , deadenylation 6 and translational repression 7 . The evolutionary conservation of miRNAs has been described 8 , as has their use of RNA polymerase II (Pol II) promoters for control of expression 9-11 . A recent study described approximately ten mammalian miRNAs created by a repetitive element transposition and subsequent transcription across two tandem, inverted repetitive elements 12 . In this study, we tested if other miRNAs possess sequences repetitive in origin, and the transcriptional requirements of a dense cluster of human miRNAs interspersed among repetitive Alu elements on chromosome 19. RESULTS miRNAs localize to repeats within the human genome We expanded prior studies by screening sequences flanking all known human miRNAs for repetitive elements (500 bp upstream and down stream) and annotating them using the Censor web server at the Genetic Information Research Institute 13 . Overall, we found that the mature sequences from B50 human miRNAs reside within repetitive elements Upstream Alus express downstream miRNAs We used degenerate primers and thermostable polymerase to first confirm that miRNAs within the C19MC are transcribed in vivo (Supplementary Table 2 online). To discern how the C19MC miRNAs are expressed, we used one of these miRNAs, miR-517a, and sequences immediately upstream, which consist of an Alu We next made reporter constructs to test whether the hairpins expressed from the miR-517a 5¢ Alu had silencing activity. These reporters (517-AS and LacZ-AS) produced Renilla luciferase transcripts with target sequences for miR-517a or shLacZ cloned into the 3¢ UTR

The microRNAs of Caenorhabditis elegans

by Lee P. Lim, Nelson C. Lau, Earl G. Weinstein, Aliaa Abdelhakim, Soraya Yekta, Matthew W. Rhoades, Christopher B. Burge, Davidp Bartel - Genes Dev , 2003
"... MicroRNAs (miRNAs) are an abundant class of tiny RNAs thought to regulate the expression of protein-coding genes in plants and animals. In the present study, we describe a computational procedure to identify miRNA genes conserved in more than one genome. Applying this program, known as MiRscan, toge ..."
Abstract - Cited by 186 (13 self) - Add to MetaCart
MicroRNAs (miRNAs) are an abundant class of tiny RNAs thought to regulate the expression of protein-coding genes in plants and animals. In the present study, we describe a computational procedure to identify miRNA genes conserved in more than one genome. Applying this program, known as MiRscan, together with molecular identification and validation methods, we have identified most of the miRNA genes in the nematode Caenorhabditis elegans. The total number of validated miRNA genes stands at 88, with no more than 35 genes remaining to be detected or validated. These 88 miRNA genes represent 48 gene families; 46 of these families (comprising 86 of the 88 genes) are conservedin Caenorhabditis briggsae, and22 families are conservedin humans. More than a thirdof the worm miRNAs, including newly identified members of the lin-4 and let-7 gene families, are differentially expressed during larval development, suggesting a role for these miRNAs in mediating larval developmental transitions. Most are present at very high steady-state levels—more than 1000 molecules per cell, with some exceeding 50,000 molecules per cell. Our census of the worm miRNAs andtheir expression patterns helps define this class of noncoding RNAs, lays the groundwork for functional studies, and provides the tools for more comprehensive analyses of miRNA genes in other species. [Keywords: miRNA; noncoding RNA; computational gene identification; Dicer] Supplemental material is available at
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Genetic and functional diversification of small RNA pathways in plants

by Zhixin Xie, Lisa K. Johansen, Adam M. Gustafson, Kristin D. Kasschau, Andrew D. Lellis, Daniel Zilberman, Steven E. Jacobsen, James C. Carrington - PLoS Biol , 2004
"... Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and g ..."
Abstract - Cited by 185 (17 self) - Add to MetaCart
Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

Human embryonic stem cells express a unique set of microRNAs.

by Mi-Ra Suh , Yoontae Lee , Jung Yeon Kim , Soo-Kyoung Kim , Sung-Hwan Moon , Ji Yeon Lee , Kwang-Yul Cha , Hyung Min Chung , Hyun Soo Yoon , Shin Yong Moon , V Narry Kim , Kye-Seong Kim - Dev. Biol. , 2004
"... Abstract Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by diffic ..."
Abstract - Cited by 106 (4 self) - Add to MetaCart
Abstract Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by difficulties in culture and by scant knowledge concerning the regulatory mechanism. Recent evidence from plants and animals indicates small RNAs of approximately 22 nucleotides (nt), collectively named microRNAs, play important roles in developmental regulation. Here we describe 36 miRNAs (from 32 stem-loops) identified by cDNA cloning in hES cells. Importantly, most of the newly cloned miRNAs are specifically expressed in hES cells and downregulated during development into embryoid bodies (EBs), while miRNAs previously reported from other human cell types are poorly expressed in hES cells. We further show that some of the ES-specific miRNA genes are highly related to each other, organized as clusters, and transcribed as polycistronic primary transcripts. These miRNA gene families have murine homologues that have similar genomic organizations and expression patterns, suggesting that they may operate key regulatory networks conserved in mammalian pluripotent stem cells. The newly identified hES-specific miRNAs may also serve as molecular markers for the early embryonic stage and for undifferentiated hES cells.
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Identification of Drosophila microRNA targets

by Er Stark, Julius Brennecke, Robert B. Russell, Stephen M. Cohen - PLoS Biol , 2003
"... MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in i ..."
Abstract - Cited by 103 (5 self) - Add to MetaCart
MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 39 UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA–target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.

by Akira Ishizuka , Mikiko C Siomi , Haruhiko Siomi - Genes Dev. , 2002
"... Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR ..."
Abstract - Cited by 102 (2 self) - Add to MetaCart
Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.
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Sequence requirements for micro RNA processing and function in human cells

by Yan Zeng, Bryan R. Cullen - RNA , 2003
"... Most eukaryotes encode a substantial number of small noncoding RNAs termed micro RNAs (miRNAs). Previously, we have demonstrated that miR-30, a 22-nucleotide human miRNA, can be processed from a longer transcript bearing the proposed miR-30 stem-loop precursor and can translationally inhibit an mRNA ..."
Abstract - Cited by 85 (7 self) - Add to MetaCart
Most eukaryotes encode a substantial number of small noncoding RNAs termed micro RNAs (miRNAs). Previously, we have demonstrated that miR-30, a 22-nucleotide human miRNA, can be processed from a longer transcript bearing the proposed miR-30 stem-loop precursor and can translationally inhibit an mRNA-bearing artificial target sites. We also demonstrated that the miR-30 precursor stem can be substituted with a heterologous stem, which can be processed to yield novel miRNAs and can block the expression of endogenous mRNAs. Here, we show that a second human miRNA, termed miR-21, can also be effectively expressed when its precursor forms part of a longer mRNA. For both miR-30 and miR-21, mature miRNA production was highly dependent on the integrity of the precursor RNA stem, although the underlying sequence had little effect. In contrast, the sequence of the terminal loop affected miRNA production only moderately. Processing of the initial, miR-30-containing transcript led to the production of not only mature miR-30 but also to the largely nuclear excision of an ∼65-nucleotide RNA that is likely to represent an important intermediate in miR-30 processing. Consistent with this hypothesis, mutations that affected mature miR-30 production inhibited expression of this miR-30 pre-miRNA to an equivalent degree. Although point mutations could block the ability of both miR-30 and miR-21 to inhibit the translation of mRNAs bearing multiple artificial miRNA target sites, single point mutations only attenuated the miRNA-mediated inhibition of genes bearing single, fully complementary targets. These results suggest that miRNAs, and the closely similar small interfering RNAs, cannot totally discriminate between RNA targets differing by a single nucleotide.
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Endogenous and silencing-associated small RNAs in plants

by Cesar Llave, Kristin D. Kasschau, Maggie A. Rector, James C. Carrington - Downloaded from http://nar.oxfordjournals.org/ at Pennsylvania State University on February 23, 2013 Acids Research , 2002
"... A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 ..."
Abstract - Cited by 72 (6 self) - Add to MetaCart
A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 sequences identified, the vast majority (90%) arose from intergenic regions, although small RNAs corresponding to predicted protein-coding genes, transposon-like sequences, and a structural RNA gene also were identified. Evidence consistent with the derivation of small RNAs of both polarities, and from highly base-paired precursors, was obtained through the identification and analysis of clusters of small RNA loci. The accumulation of specific small RNAs was regulated developmentally. We propose that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.
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