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Ando H, Hirose M, Kurosawa G, et al. Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells. Sci Rep. 2017;7(1):12642doi: 10.1038/s41598-017-12879-2.
Ando, H., Hirose, M., Kurosawa, G., Impey, S., & Mikoshiba, K. (2017). Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells. Scientific reports, 7(1), 12642. https://doi.org/10.1038/s41598-017-12879-2
Ando, Hideaki, et al. "Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells." Scientific reports vol. 7,1 (2017): 12642. doi: https://doi.org/10.1038/s41598-017-12879-2
Ando H, Hirose M, Kurosawa G, Impey S, Mikoshiba K. Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells. Sci Rep. 2017 Oct 03;7(1):12642. doi: 10.1038/s41598-017-12879-2. PMID: 28974737; PMCID: PMC5626736.
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Sci Rep. 2017 Oct 03;7(1):12642. doi: 10.1038/s41598-017-12879-2.

Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells.

Scientific reports

Hideaki Ando, Matsumi Hirose, Gen Kurosawa, Soren Impey, Katsuhiko Mikoshiba

Affiliations

  1. Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako, Saitama, 351-0198, Japan. [email protected].
  2. Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako, Saitama, 351-0198, Japan.
  3. Theoretical Biology Laboratory, RIKEN, Wako, Saitama, 351-0198, Japan.
  4. Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR, 97239, USA.

PMID: 28974737 PMCID: PMC5626736 DOI: 10.1038/s41598-017-12879-2

Abstract

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.

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