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      Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells

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      PLoS ONE
      Public Library of Science

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          Abstract

          MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1 α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1 α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level.

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          Most cited references51

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          Origins and Mechanisms of miRNAs and siRNAs.

          Over the last decade, approximately 20-30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs--short interfering RNAs (siRNAs) and microRNAs (miRNAs)--act in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment.
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            Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.

            MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.
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              Prediction of mammalian microRNA targets.

              MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2016
                6 May 2016
                : 11
                : 5
                : e0155177
                Affiliations
                [001]Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Baden-Württemberg, Germany
                IPMC, CNRS UMR 7275 UNS, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: HLS PAN. Performed the experiments: HLS. Analyzed the data: HLS PAN. Contributed reagents/materials/analysis tools: HLS PAN. Wrote the paper: HLS PAN.

                Author information
                http://orcid.org/0000-0003-4939-3418
                Article
                PONE-D-16-09196
                10.1371/journal.pone.0155177
                4859538
                27152616
                77f94d18-ed00-4276-bbe3-915a53a7f16b
                © 2016 Sladitschek, Neveu

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 March 2016
                : 3 April 2016
                Page count
                Figures: 6, Tables: 1, Pages: 16
                Funding
                The study was funded by the European Molecular Biology Laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                MicroRNAs
                Biology and Life Sciences
                Developmental Biology
                Cell Differentiation
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Spectrophotometry
                Cytophotometry
                Flow Cytometry
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Cell Potency
                Pluripotency
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Messenger RNA
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Double stranded RNA
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Enhancer Elements
                Custom metadata
                All relevant data are within the paper.

                Uncategorized
                Uncategorized

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