Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells
Autor: | Matsumi Hirose, Hideaki Ando, Katsuhiko Mikoshiba, Soren Impey, Gen Kurosawa |
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Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
RNA Stability Kinetics lcsh:Medicine Biology Time-Lapse Imaging Article 03 medical and health sciences 0302 clinical medicine Transcription (biology) microRNA Gene silencing Humans lcsh:Science Regulation of gene expression Multidisciplinary lcsh:R Cell biology MicroRNAs 030104 developmental biology Gene Expression Regulation Proteome lcsh:Q Single-Cell Analysis MiRNA biogenesis 030217 neurology & neurosurgery Biogenesis |
Zdroj: | Scientific Reports Scientific Reports, Vol 7, Iss 1, Pp 1-16 (2017) |
ISSN: | 2045-2322 |
Popis: | MicroRNAs (miRNAs) are small, non-coding RNAs that play critical roles in the post-transcriptional regulation of gene expression. Although the molecular mechanisms of the biogenesis and activation of miRNA have been extensively studied, the details of their kinetics within individual living cells remain largely unknown. We developed a novel method for time-lapse imaging of the rapid dynamics of miRNA activity in living cells using destabilized fluorescent proteins (dsFPs). Real-time monitoring of dsFP-based miRNA sensors revealed the duration necessary for miRNA biogenesis to occur, from primary miRNA transcription to mature miRNA activation, at single-cell resolution. Mathematical modeling, which included the decay kinetics of the fluorescence of the miRNA sensors, demonstrated that miRNAs induce translational repression depending on their complementarity with targets. We also developed a dual-color imaging system, and demonstrated that miR-9-5p and miR-9-3p were produced and activated from a common hairpin precursor with similar kinetics, in single cells. Furthermore, a dsFP-based miR-132 sensor revealed the rapid kinetics of miR-132 activation in cortical neurons under physiological conditions. The timescale of miRNA biogenesis and activation is much shorter than the median half-lives of the proteome, suggesting that the degradation rates of miRNA target proteins are the dominant rate-limiting factors for miRNA-mediated gene silencing. |
Databáze: | OpenAIRE |
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