Inducible expression of coding and inhibitory RNAs from retargetable genomic loci

Weidenfeld, Ina; Gossen, Manfred; Löw, Rainer; Kentner, David; Berger, Stefan; Görlich, Dirk; Bartsch, Dušan; Bujard, Hermann; Shi, Kai

doi:10.1093/nar/gkp108

Cited by 76 publications

(91 citation statements)

References 34 publications

(61 reference statements)

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“…the same locus using a recently published strategy [20]. Indeed, on induction, expression of the control FL reporter was virtually identical in independent cell lines ( Fig 1B; supplementary Fig S1 online).…”

Section: Results and Discussion Stable Cell Lines Expressing Induciblmentioning

confidence: 70%

Kinetic analysis reveals successive steps leading to miRNA‐mediated silencing in mammalian cells

2012

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MicroRNAs (miRNAs) regulate most cellular functions, acting by posttranscriptionally repressing numerous eukaryotic mRNAs. They lead to translational repression, deadenylation and degradation of their target mRNAs. Yet, the relative contributions of these effects are controversial and little is known about the sequence of events occurring during the miRNA-induced response. Using stable human cell lines expressing inducible reporters, we found that translational repression is the dominant effect of miRNAs on newly synthesized targets. This step is followed by mRNA deadenylation and decay, which is the dominant effect at steady state. Our findings have important implications for understanding the mechanism of silencing and reconcile seemingly contradictory data.

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Section: Results and Discussion Stable Cell Lines Expressing Induciblmentioning

confidence: 70%

Kinetic analysis reveals successive steps leading to miRNA‐mediated silencing in mammalian cells

2012

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“…Cells were lysed 24 h after transfection. For TNRC6 rescue experiments, HeLa cells stably expressing Tet-On machinery 36 were transfected using attractene reagent (Qiagen). Per well of the 96-well plate, transfection mixtures contained 10 ng of the let-7 reporter plasmid, increasing amounts of NHA-TNRC6A or its point mutants (20, 60 and 180 ng), and either siRNAs specific to TNRC6A, B and C (5′-GCCUAAUCUCCGUGCUCAATT-3′, 5′-GGCCUUGUAUUGCCAGCAATT-3′ and 5′-GCAUUAAGUGCUAAACAAATT-3′ (Microsynth; sequences represent sense strands), 0.53 pmol each; or 1.6 pmol AllStars siRNA negative control (Qiagen).…”

Section: Cell Culture Transfections Rnai and Luciferase Assaysmentioning

confidence: 99%

miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

Chekulaeva

Mathys

Zipprich

et al. 2011

Nat Struct Mol Biol

324

449

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AbstractmiRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/ Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR 4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR 4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR 4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNA s, including inhibiting translation independently of deadenylation.MicroRNAs (miRNAs) are small, ~21-nt-long RNAs that post-transcriptionally regulate gene expression in eukaryotes. In animals, miRNAs bind to partially complementary sites in mRNAs, leading to translational repression and mRNA deadenylation and degradation [1][2][3][4] . miRNAs function as part of ribonucleoprotein complexes, miRNPs, with Argonaute (AGO) and GW182 family proteins being the crucial components. GW182s interact directly with AGO proteins and function downstream as effectors mediating mRNA repression. Hence, understanding the function of GW182 proteins is critical for understanding miRNAmediated repression.GW182 functional regions have been mapped in D. melanogaster and mammalian proteins. In D. melanogaster, three regions were found to repress tethered mRNA to a similar extent 5

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“…Stable HeLa cell lines expressing enhanced yellow fluorescent protein (EYFP), super green fluorescent protein (SGFP), enhanced green fluorescent protein (EGFP), Katushka, 29 or Cardinal 30 red fluorescent protein were generated and characterized as described by Weidenfeld et al 26 In brief, HeLa Tet-ON advanced cells (line EM2-11ht) were stably transfected with DNA coding for the fluorescent proteins under control of a tetracycline/doxycycline inducible promoter (Ptet). The gene of interest was stably integrated into a defined genomic locus (5q31), which allows tight regulation of the doxycycline-inducible expression.…”

Section: Cell Culturementioning

confidence: 99%

“…To this purpose, we made use of a Tet-ON conditional gene expression system 26 where five FPs were cloned in stable cell lines within the same genetic locus, thus resulting in identical mRNA expression levels. Since promoter activity was equal, it follows that variability in fluorescence emission intensity could be directly related to the activity of each FP (including brightness, stability, cell toxicity, and so on).…”

Section: Introductionmentioning

confidence: 99%

Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

et al. 2016

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Abstract. Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.

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Inducible expression of coding and inhibitory RNAs from retargetable genomic loci

Cited by 76 publications

References 34 publications

Kinetic analysis reveals successive steps leading to miRNA‐mediated silencing in mammalian cells

Kinetic analysis reveals successive steps leading to miRNA‐mediated silencing in mammalian cells

miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

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