Dataset Information

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.

ABSTRACT: Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of "highly expressed equals high repression".

SUBMITTER: Lemus-Diaz N

PROVIDER: S-EPMC5364550 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.

Lemus-Diaz Nicolas N Böker Kai O KO Rodriguez-Polo Ignacio I Mitter Michael M Preis Jasmin J Arlt Maximilian M Gruber Jens J

Scientific reports 20170324

Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling ...[more]

PMID: 28338079

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Project description:BACKGROUND:Fluorescent reporter genes have become widely used for monitoring gene expression in living cells. When a microbial strain carrying a reporter gene is grown in a microplate reader, the fluorescence and the absorbance (optical density) of the culture can be automatically measured every few minutes in a highly parallelized way. The extraction of useful information from the resulting large amounts of data is not easy to achieve, because the fluorescence and absorbance measurements are only indirectly related to promoter activities and protein concentrations, requiring mathematical models of the expression of reporter genes for their interpretation. Although the principles of the analysis of reporter gene data are well-established today, there is a lack of general-purpose bioinformatics tools based on generic measurement models and sound inference procedures. This has motivated the development of WellInverter, a web application based on well-known methods for regularized linear inversion. RESULTS:We present a new version of WellInverter that considerably improves the performance and usability of the original application. In particular, we have put in place a parallel computing architecture with a load balancer to distribute analysis queries over several back-end servers, we have completely redesigned the graphical user interface to better support the different analysis steps, and we have developed a plug-in system for the parsing of data files produced by microplate readers from different manufacturers. We illustrate the functioning of WellInverter by analyzing data of the expression of a fluorescent reporter gene controlled by a phage promoter in growing Escherichia coli populations. We show that the expression pattern in different growth media, supporting different growth rates, corresponds to the pattern expected for a constitutive gene. CONCLUSIONS:The new version of WellInverter is a robust, easy-to-use and scalable web application, which has been deployed on two publicly accessible web servers and which can also be installed locally. A demo version of the application with two sample datasets is available on-line.

Dataset Information

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.

Publications

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.

Similar Datasets

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