Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system
Open accessAbstract
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".
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Low expression but functional miRNA may synergistically increase RNAi activity on a subset of target mRNAs, thereby effectively enhancing the concentration of cognate miRNAs for specific mRNAs.
“In this way, low expression but functional miRNA may synergistically increase RNAi activity on a subset of target mRNAs, thereby effectively enhancing the concentration of cognate miRNAs for specific mRNAs (101, 102).”
From Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation (2018) · cites this paper as [102]
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- Authors
- Lemus-Diaz, N., Böker, K. O., Rodriguez-Polo, I., Mitter, M., Preis, J., Arlt, M., & Gruber, J.
- Affiliations
- German Primate Center (DE)
- Corresponding author
- Gruber, Jens
- Venue
- Scientific Reports
- Published
- 2017-03-24
- Volume
- 7
- Issue
- 1
- Pages
- 45197-45197
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC5364550
- OpenAlex
- W2603866810
- Cited by
- 18
- References
- 67
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2022: 3 · 2021: 5 · 2020: 2 · 2019: 2 · 2018: 3
- FWCI
- 0.64
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion, cc-by)
- GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
- Funders
- Deutsche Forschungsgemeinschaft (GSC 226/2, GSC 226)
- Leibniz-Gemeinschaft
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Keywords
- microRNA, gene repression, single-cell analysis, fluorescent reporter assay, miRNAome, gene expression regulation
- MeSH terms
- HeLa Cells, Humans, Microscopy, Fluorescence, Transcription, Genetic, Down-Regulation, MicroRNAs, HEK293 Cells, Single-Cell Analysis
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