378 research outputs found

    MiR-10 Represses HoxB1a and HoxB3a in Zebrafish

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    BACKGROUND: The Hox genes are involved in patterning the anterior-posterior axis. In addition to the protein coding Hox genes, the miR-10, miR-196 and miR-615 families of microRNA genes are conserved within the vertebrate Hox clusters. The members of the miR-10 family are located at positions associated with Hox-4 paralogues. No function is yet known for this microRNA family but the genomic positions of its members suggest a role in anterior-posterior patterning. METHODOLOGY/PRINCIPAL FINDINGS: Using sensor constructs, overexpression and morpholino knockdown, we show in Zebrafish that miR-10 targets HoxB1a and HoxB3a and synergizes with HoxB4 in the repression of these target genes. Overexpression of miR-10 also induces specific phenotypes related to the loss of function of these targets. HoxB1a and HoxB3a have a dominant hindbrain expression domain anterior to that of miR-10 but overlap in a weaker expression domain in the spinal cord. In this latter domain, miR-10 knockdown results in upregulation of the target genes. In the case of a HoxB3a splice variant that includes miR-10c within its primary transcript, we show that the microRNA acts in an autoregulatory fashion. CONCLUSIONS/SIGNIFICANCE: We find that miR-10 acts to repress HoxB1a and HoxB3a within the spinal cord and show that this repression works cooperatively with HoxB4. As with the previously described interactions between miR-196 and HoxA7 and Hox-8 paralogues, the target genes are located in close proximity to the microRNA. We present a model in which we postulate a link between the clustering of Hox genes and post-transcriptional gene regulation. We speculate that the high density of transcription units and enhancers within the Hox clusters places constraints on the precision of the transcriptional control that can be achieved within these clusters and requires the involvement of post-transcriptional gene silencing to define functional domains of genes appropriately

    The Impact of miRNA Target Sites in Coding Sequences and in 3β€²UTRs

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    Animal miRNAs are a large class of small regulatory RNAs that are known to directly and negatively regulate the expression of a large fraction of all protein encoding genes. The identification and characterization of miRNA targets is thus a fundamental problem in biology. miRNAs regulate target genes by binding to 3β€² untranslated regions (3β€²UTRs) of target mRNAs, and multiple binding sites for the same miRNA in 3β€²UTRs can strongly enhance the degree of regulation. Recent experiments have demonstrated that a large fraction of miRNA binding sites reside in coding sequences. Overall, miRNA binding sites in coding regions were shown to mediate smaller regulation than 3β€²UTR binding. However, possible interactions between target sites in coding sequences and 3β€²UTRs have not been studied. Using transcriptomics and proteomics data of ten miRNA mis-expression experiments as well as transcriptome-wide experimentally identified miRNA target sites, we found that mRNA and protein expression of genes containing target sites both in coding regions and 3β€²UTRs were in general mildly but significantly more regulated than those containing target sites in 3β€²UTRs only. These effects were stronger for conserved target sites of length 7–8 nt in coding regions compared to non-conserved sites. Combined with our other finding that miRNA target sites in coding regions are under negative selection, our results shed light on the functional importance of miRNA targeting in coding regions

    Pre-processing and differential expression analysis of Agilent microRNA arrays using the AgiMicroRna Bioconductor library

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    <p>Abstract</p> <p>Background</p> <p>The main research tool for identifying microRNAs involved in specific cellular processes is gene expression profiling using microarray technology. Agilent is one of the major producers of microRNA arrays, and microarray data are commonly analyzed by using R and the functions and packages collected in the Bioconductor project. However, an analytical package that integrates the specific characteristics of microRNA Agilent arrays has been lacking.</p> <p>Results</p> <p>This report presents the new bioinformatic tool <it>AgiMicroRNA </it>for the pre-processing and differential expression analysis of Agilent microRNA array data. The software is implemented in the open-source statistical scripting language R and is integrated in the Bioconductor project (<url>http://www.bioconductor.org</url>) under the GPL license. For the pre-processing of the microRNA signal, <it>AgiMicroRNA </it>incorporates the <it>robust multiarray average algorithm</it>, a method that produces a summary measure of the microRNA expression using a linear model that takes into account the probe affinity effect. To obtain a normalized microRNA signal useful for the statistical analysis, <it>AgiMicroRna </it>offers the possibility of employing either the processed signal estimated by the <it>robust multiarray average algorithm </it>or the processed signal produced by the Agilent image analysis software. The <it>AgiMicroRNA </it>package also incorporates different graphical utilities to assess the quality of the data. <it>AgiMicroRna </it>uses the linear model features implemented in the <it>limma </it>package to assess the differential expression between different experimental conditions and provides links to the <it>miRBase </it>for those microRNAs that have been declared as significant in the statistical analysis.</p> <p>Conclusions</p> <p><it>AgiMicroRna </it>is a rational collection of Bioconductor functions that have been wrapped into specific functions in order to ease and systematize the pre-processing and statistical analysis of Agilent microRNA data. The development of this package contributes to the Bioconductor project filling the gap in microRNA array data analysis.</p

    miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

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    <p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.</p> <p>Results</p> <p>Here, we present the miR-Q assay as a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as miRNA molecules. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM miRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays. Using this approach, we quantified the expression of let-7b in different human cell lines as well as miR-145 and miR-21 expression in porcine intestinal samples.</p> <p>Conclusion</p> <p>miR-Q is a cost-effective and highly specific approach, which neither requires the use of fluorochromic probes, nor Locked Nucleic Acid (LNA)-modified oligonucleotides. Moreover, it provides a remarkable increase in specificity and simplified detection of small RNAs.</p

    Detection and Verification of Mammalian Mirtrons by Northern Blotting

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    microRNAs (miRNAs) have vital roles in regulating gene expressionβ€”contributing to major diseases like cancer and heart disease. Over the last decade, thousands of miRNAs have been discovered through high throughput sequencing-based annotation. Different classes have been described, as well as a great dynamic range of expression levels. While sequencing approaches provide insight into biogenesis and allow confident identification, there is a need for additional methods for validation and characterization. Northern blotting was one of the first techniques used for studying miRNAs, and remains one of the most valuable as it avoids enzymatic manipulation of miRNA transcripts. Blotting can also provide insight into biogenesis by revealing RNA processing intermediates. Compared to sequencing, however, northern blotting is a relatively insensitive technology. This creates a challenge for detecting low expressed miRNAs, particularly those produced by inefficient, non-canonical pathways. In this chapter, we describe a strategy to study such miRNAs by northern blotting that involves ectopic expression of both miRNAs and miRNA-binding Argonaute (Ago) proteins. Through use of epitope tags, this strategy also provides a convenient method for verification of small RNA competency to be loaded into regulatory complexes

    Analysis of MicroRNA Expression in Embryonic Developmental Toxicity Induced by MC-RR

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    As cynobacterial blooms frequently occur in fresh waters throughout the world, microcystins (MCs) have caused serious damage to both wildlife and human health. MCs are known to have developmental toxicity, however, the possible molecular mechanism is largely unknown. This is the first toxicological study to integrate post-transcriptomic, proteomic and bioinformatics analysis to explore molecular mechanisms for developmental toxicity of MCs in zebrafish. After being microinjected directly into embryos, MC-RR dose-dependently decreased survival rates and increased malformation rates of embryos, causing various embryo abnormalities including loss of vascular integrity and hemorrhage. Expressions of 31 microRNAs (miRNAs) and 78 proteins were significantly affected at 72 hours post-fertilisation (hpf). Expressions of miR-430 and miR-125 families were also significantly changed. The altered expressions of miR-31 and miR-126 were likely responsible for the loss of vascular integrity. MC-RR significantly reduced the expressions of a number of proteins involved in energy metabolism, cell division, protein synthesis, cytoskeleton maintenance, response to stress and DNA replication. Bioinformatics analysis shows that several aberrantly expressed miRNAs and proteins (involved in various molecular pathways) were predicted to be potential MC-responsive miRNA-target pairs, and that their aberrant expressions should be the possible molecular mechanisms for the various developmental defects caused by MC-RR

    Plasma MicroRNA Profiles in Rat Models of Hepatocellular Injury, Cholestasis, and Steatosis

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    MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121–317 miRNAs) were increased over 2-fold and the levels of a small number of miRNAs (6–35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and identified the miRNAs that could be specific and sensitive biomarkers of liver injury

    Three Drosophila Hox Complex microRNAs Do Not Have Major Effects on Expression of Evolutionarily Conserved Hox Gene Targets during Embryogenesis

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    The discovery of microRNAs has resulted in a major expansion of the number of molecules known to be involved in gene regulation. Elucidating the functions of animal microRNAs has posed a significant challenge as their target interactions with messenger RNAs do not adhere to simple rules. Of the thousands of known animal microRNAs, relatively few microRNA:messenger RNA regulatory interactions have been biologically validated in an normal organismal context. Here we present evidence that three microRNAs from the Hox complex in Drosophila (miR-10-5p, miR-10-3p, miR-iab-4-5p) do not have significant effects during embryogenesis on the expression of Hox genes that contain high confidence microRNAs target sites in the 3β€² untranslated regions of their messenger RNAs. This is significant, in that it suggests that many predicted microRNA-target interactions may not be biologically relevant, or that the outcomes of these interactions may be so subtle that mutants may only show phenotypes in specific contexts, such as in environmental stress conditions, or in combinations with other microRNA mutations

    RNA deep sequencing reveals differential MicroRNA expression during development of sea urchin and sea star

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    microRNAs (miRNAs) are small (20-23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. Β© 2011 Kadri et al

    Down-Regulation of miR-92 in Human Plasma Is a Novel Marker for Acute Leukemia Patients

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    BACKGROUND: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma. METHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body. CONCLUSIONS/SIGNIFICANCE: The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia
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